 |
 Previous Article | Next Article 
The Journal of Neuroscience, May 15, 2000, 20(10):3705-3713
Wild-Type Huntingtin Protects from Apoptosis Upstream of
Caspase-3
Dorotea
Rigamonti1,
Johannes H.
Bauer2,
Claudio
De-Fraja1,
Luciano
Conti1,
Simonetta
Sipione1,
Clara
Sciorati3,
Emilio
Clementi3, 4,
Abigail
Hackam5,
Michael R.
Hayden5,
Yong
Li2,
Jillian K.
Cooper6,
Christopher A.
Ross6,
Stefano
Govoni7,
Claudius
Vincenz2, and
Elena
Cattaneo1
1 Institute of Pharmacological Sciences, University of
Milano, 20133 Milano, Italy, 2 Department of Pathology,
University of Michigan, Ann Arbor, Michigan 48109-0726, 3 Department of Biotechnology, San Raffaele
Hospital, Milano, Italy, 4 Department of Pharmacobiology,
University of Calabria, 87036 Rende, Italy, 5 University of
British Columbia, V52 4H4 Vancouver, British Columbia, Canada,
6 Department of Psychiatry, Johns Hopkins University,
Baltimore, Maryland 21025, and 7 Institute of Pharmacology,
University of Pavia, 27100 Pavia, Italy
 |
ABSTRACT |
Expansion of a polyglutamine sequence in the N terminus of
huntingtin is the gain-of-function event that causes Huntington's disease. This mutation affects primarily the medium-size spiny neurons
of the striatum. Huntingtin is expressed in many neuronal and
non-neuronal cell types, implying a more general function for the
wild-type protein. Here we report that wild-type huntingtin acts by
protecting CNS cells from a variety of apoptotic stimuli, including serum withdrawal, death receptors, and pro-apoptotic Bcl-2
homologs. This protection may take place at the level of caspase-9
activation. The full-length protein also modulates the toxicity of the
poly-Q expansion. Cells expressing full-length mutant protein are
susceptible to fewer death stimuli than cells expressing truncated
mutant huntingtin.
Key words:
huntingtin; CAG; caspases; survival; CNS cells; Huntington's disease
| |
INTRODUCTION |
Huntington's disease (HD) is an
autosomal neurodegenerative disorder caused by a (CAG)n expansion in
the gene IT15 (Huntington's disease Collaborative Research Group,
1993 ). This trinucleotide expansion encodes for a polyglutamine
(QN) stretch in the encoded protein, huntingtin
(Htt), resulting in progressive neuronal death with clinical symptoms
of chorea and dementia (Ross et al., 1997). Studies on the cellular
localization of the mRNA and protein have revealed that wild-type (wt)
and mutated (mu) Htt are expressed throughout the nervous system as
well as in non-neuronal cells (Sharp and Ross, 1996). Despite this
widespread distribution, only specific neurons in the striatum and
cortex are affected in HD (Vonsattel et al., 1985; Kowall et al.,
1987). The cytotoxicity of the mu protein is believed to be caused by a
gain-of-function effect induced by the presence of the expanded CAG
(for review, see MacDonald and Gusella, 1996).
The molecular mechanism by which the poly-Q expansion triggers cell
death is not understood (Reddy et al., 1999). Intracellular inclusions
may be involved, but it is not clear whether they have a direct role in
pathogenesis or are involved in progression (Davies et al., 1997; Di
Figlia et al., 1997; Saudou et al., 1998) (for review, see Kim and
Tanzi, 1998; Sisodia, 1998; Gutekunst et al., 1999; Hodgson et al.,
1999). Proteolytic cleavage has been implicated by the findings of
N-terminal fragments in inclusions and the fact that Htt is a caspase
substrate and is actively cleaved during apoptosis in cultured cells
(Goldberg et al., 1996; Davies et al., 1997; DiFiglia et al., 1997;
Wellington et al., 1998; Kim et al., 1999). Experiments performed in
primary striatal neurons showed that the poly-Q cytotoxicity can be
inhibited by the anti-apoptotic protein Bcl-Xl and caspase inhibitors
(Saudou et al., 1998). Involvement of caspases is also indicated by the
observation that dominant negative caspase-1 delays disease progression
in transgenic mice (Ona et al., 1999). Studies using an isolated poly-Q
sequence have suggested that the first pathogenic event is the
aggregation and activation of caspase-8 (Sanchez et al., 1999).
Finally, activation of caspase-3 and caspase-9 was reported in HD
lymphoblasts (Sawa et al., 1999).
Much less is known about the physiological roles of wtHtt. Mice with a
targeted disruption of the Htt gene die at embryonic day 7.5 (Duyao et
al., 1995; Nasir et al., 1995; Zeitlin et al., 1995). Moreover,
analysis of mice expressing <50% of wtHtt showed extensive brain
abnormalities and died shortly after birth (White et al., 1997). These
data suggest that Htt is an important factor during embryonic
development and that its role is not restricted to the nervous system.
To assess the function of wt and muHtt in neural cells, we developed a
cell culture system composed of clonal striatal-derived cells
overexpressing the wt or muHtt protein. We found that wtHtt has a role
as a pro-survival molecule and acts upstream of caspase-3 and
downstream of pro-apoptotic Bcl-2 proteins. We also found that toxicity
of the poly-Q expansion is modulated by the length of Htt backbone.
| |
MATERIALS AND METHODS |
Constructs and transfections. FLwt [FL-23Q] and
FLmu [FL-82Q] (Cooper, 1998), N548wt [nt1955-15] and N548mu
[nt1955-128] (Hackam, 1998), myc-tagged N63wt [N63-18Q] and
N63mu [N63-82Q] (Cooper, 1998) were subcloned into pLXSP vector or
cotransfected with pLXSP as previously described (Cattaneo et al.,
1996a). Briefly, ST14A cells were grown routinely at 33°C in
the presence of DMEM supplemented with 10% fetal bovine serum
as described (Cattaneo and Conti, 1998). Ten micrograms of cDNA were
transfected using calcium phosphate or lipofectamine (Life
Technologies) (Cattaneo et al., 1996b). Resistant clones
were selected with puromycin 3 µg/ml, expanded, and cryopreserved.
Transfection with the empty plasmid was also performed. For each
construct, ~20 colonies were isolated with cloning rings and analyzed.
Protein expression assay. Expression of the cDNAs was
analyzed by Western Blot (WB) using antibody MAB2166 (dilution 1:2000; Chemicon, Temecula, CA), BKP1 (dilution 1:50), Ab1 (1 µg/ml, kindly provided by M. DiFiglia), and Ap194 (dilution 1:1000; kindly provided by A. Sharp). For N63wt and N63mu cDNAs immunoprecipitation with an
anti-myc antibody (9E10, dilution 1:100; Calbiochem, La Jolla, CA) was
performed after metabolic labeling of the cells. A total of 50 µg of
proteins was loaded in each lane. To control for loading of the gels,
an anti-actin antibody (Sigma, St. Louis, MO; 1:2000) was used. All
assays were performed on cells that have been cultured up to the 25th passage.
Cell counting, MTT assay, and BrDU measurement. Cells were
plated in triplicates into six well plates at a density of 2 × 105/well. After 8 hr incubation at 33°C
the cultures were washed with HBSS, the medium was replaced with
serum-deprived medium (SDM; composition: F-12/DMEM, 5 mg/l insulin, 100 mg/l transferrin, 20 nM progesterone, 30 nM
selenite, 60 mM putrescine, 2 mM glutamine, 0.11 gm/l sodium pyruvate, 3.7 gm/l sodium bicarbonate, and 3.9 gm/l
HEPES) and then incubated at 39°C. At the times indicated, cells were
trypsinized, resuspended in 20 ml Isoton, and counted using a Coulter
Counter machine (ZM; Coulter Instruments). For MTT assays, cells
were exposed to
3-[4.5-dimethylthiazol-2-phenyl]-2.5-diphenyl-tetrazolium bromide, and release of formazan from mitochondria was quantified at
560 nm using an ELISA plate reader. 3-Nitro-propionic acid was obtained
from Sigma.
For quantitation of the mitotic events, a 6 hr pulse of
bromodeoxyuridine (BrDU; 10 µM, Sigma) was given to the
cells (Cattaneo et al., 1994). At the end of incubation, cells were
rinsed, fixed for 15 min in 4% paraformaldehyde, permeabilized with
0.5% Triton X-100 for 5 min, and then incubated with anti-BrDU
antibody (Becton Dickinson, Mountain View, CA; 1:50) (Magrassi et al.,
1998).
Secondary FITC conjugated antibody to (Sigma) was used at 1:200. The
cells were viewed with a Zeiss (Axiovert) microscope, and positive
cells were scored manually.
DNA fragmentation and caspase activity. Cells were harvested
in 10 mM Tris, pH 8, 20 mM EDTA, and 2% Triton
X-100. Low molecular weight DNA was extracted by phenol/chloroform
extractions and DNA precipitated with isopropanol. DNA fragmentation
was detected on a 1% agarose gel.
Measurement of caspase-3 activity: samples of 1-2 × 106 cells were rinsed in cold PBS and
lysed in a buffer containing 25 mM HEPES, pH 7.5, 5 mM EDTA, 1 mM EGTA, 5 mM
MgCl2, 5 mM dithiothreitol, 1%
3-[-(3-cholamidopropyl)dimethylammonio]-1-propanesulfonicacid (CHAPS), 10 µg/ml pepstatin and leupeptin, and 1 mM PMSF. The cell lysates were centrifuged (for 3 min at
5000 rpm), and the supernatants stored at  80°C. Lysates were
incubated at 37°C in a buffer containing 25 mM HEPES, pH
7.5, 10% sucrose, 0.1% CHAPS, and 1 mM DTT supplemented
with Ac-DEVD-7-amino-4-methylcoumarin (amc) (50 µM). The
increase of fluorescence after the cleavage of the fluorogenic amc
moiety was monitored and then quantified in a LS50 Perkin-Elmer
(Emeryville, CA) fluorimeter (excitation, 380 nm; emission, 460 nm).
Protein content was assayed by the bicinchoninic acid procedure. For
quantitation, standard curves using increasing concentrations of amc
moiety were performed in parallel.
Constitutively active caspase-3 (Srinivasula et al., 1998) or pLXSP
plasmid (as a control) were cotransfected with the EGFP-N1 plasmid
(Clontech, Palo Alto, CA) in the ratio 10:1. Cell viability was
determined 30 hr after transfection by counting the ratio of
green-stained cells (EGFP-positive) versus total number of cells.
TUNEL assay. 2-3 × 105
cells were transfected with 250 ng of pCMV-EGFP and 1 µg of the
indicated plasmids (0.4 µg for Bik and Bak). Expression from the
transiently transfected genes was confirmed by WB and found to range
between 10 and 15 times above control. After 24 hr the cells and their
supernatant were harvested, and terminal transferase terminal
deoxynucleotidyl transferase-mediated biotinylated UTP nick end
labeling (TUNEL) was essentially performed as described previously,
using phycoerythrin (PE)-conjugated avidin (Gorczyca et
al., 1993). Stained cells were analyzed by dual color FACS, collecting
at least 10,000 events. The number of TUNEL-positive cells was
estimated by gating on the EGFP-expressing cells, excluding  95% of
untransfected cells. Transfection efficiencies were ~30-40%. The PE
gate was set using vector-only transfected cells, excluding 95% of
unstained cells. Background death was introduced by the transfection
procedure and varied between individual experiments, but was constant
within any given experiment. Therefore, background death was estimated
by transfection of control protein 14-3-3 and subsequently subtracted
from the data.
Statistical analysis. Data reported in Figures 3, 4, and 6
and in Results were compared using the one-way ANOVA test (a
p value < 0.05 was considered significant).
Statistical analysis for data reported in Figure 7 is nonparametric,
and SD derives from data obtained in three different experiments.
| |
RESULTS |
Expression of wt and muHtt in ST14A striatal cells
Conditionally immortalized ST14A cells were stably transfected
with full-length wt or poly-Q expanded Htt constructs (FLwt and
FLmu, respectively) or with the Htt truncations (N548wt, N548mu, N63wt, and N63mu), as outlined in Figure
1A.
View larger version (39K):
[in this window]
[in a new window]
|
Figure 1.
Expression of wt and muHtt in stable cell clones.
A, Outline of the Htt constructs used and abbreviation
of the stable ST14A subclones obtained. B, Expression of
wt and muHtt in stable cell clones. Western blot analysis on lysates
from clones expressing FLwt (9-25 clone), FLmu (10-6 clone), N548wt
(7-12 clone), N548mu (8-2 clone), N63wt (13-5 clone), and N63mu
(14-2 clone). The FL and 548aa-truncation were detected with MAB2166
after electrophoresis on a 6 or 10% SDS-PAGE, respectively. The
shorter 63aa protein was revealed by an anti-myc antibody after running
the samples on a 15% SDS-PAGE. Each blot includes a lane with lysates
from parental ST14A cells. Native huntingtin in the cells is detectable
at longer exposures. C, Lysates from 7-12 and 8-2
clones were analyzed for protein expression after 5, 10, and 25 passages in vitro. Equal loading was confirmed by
reacting the membrane with an anti-actin antibody.
|
|
Expression of the exogenous proteins in the different subclones was
confirmed by Western blot analysis (Fig. 1B) and
immunocytochemistry (data not shown) using antibody MAB2166 raised
against aa 181-810 of the Htt protein. As shown, a band of 340 kDa is
visible on a 6% SDS-PAGE in the FLwt and FLmu clones. Similarly, a 72 kDa band is present in N548wt clones, whereas N548mu shows a 115 kDa band. This decreased electrophoretic mobility is attributable to the
expanded CAG. Identical results were obtained with Ab1, BKP1, and AP194
antibodies (data not shown). Because the N63 constructs are myc-tagged,
their expression was confirmed by an anti-myc antibody (Fig.
1B).
Size and stability of the exogenously expressed proteins over time was
confirmed on lysates obtained from cells at the
4th, 12th and
25th passages. A representative Western
blot performed on lysates from N548wt and N548mu cells is shown in
Figure 1C. As visible, the intensity of the immunoreactive
band in cells bearing the wild-type or mutated protein did not decline
with time in vitro, up to the
25th passage. In addition, the size of the
immunoreactive band remained the same, indicating that the poly-Q
repeat is stably replicated in ST14A cells.
Wt and muHtt differentially affect cell viability
ST14A cells were derived from the embryonic striatum via
retroviral transduction of the temperature-sensitive version of the Large-T Antigen (Cattaneo et al., 1994; Cattaneo and Conti, 1998). ST14A cells grow in 10% serum at the permissive temperature of 33°C
(Fig. 2a) with a doubling time
of 36 hr (Cattaneo et al., 1996a). Shifting to the nonpermissive
temperature of 39°C leads to degradation of the Large-T Antigen, with
consequent block of cell proliferation and reversal of the phenotype
back to that of a differentiating neuroblast. This was better
demonstrated in in vivo transplantation studies (Cattaneo et
al., 1994; Lundberg et al., 1997; Benedetti et al., 2000). When ST14A
cells were exposed in vitro to 39°C in serum-deprived
medium (SDM), cells changed morphology but also their viability
decreased with time in a highly reproducible manner (Fig.
2b). In these conditions, increased cell death was observed
in cells expressing the N548mu construct (Fig. 2d). In sharp
contrast, cells overexpressing the N548wt cDNA remained viable in the
same culturing conditions (Fig. 2f).
View larger version (165K):
[in this window]
[in a new window]
|
Figure 2.
Morphology and confluency of parental ST14A
(a, b), N548mu (8-2 clone) (c, d), and
N548wt (7-12 clone) (e, f) cells in regular
passaging conditions (i.e., at 33°C in serum; a, c, e)
and after exposure to serum-deprived medium at 39°C for 48 hr
(b, d, f). N548wt evokes increased cell survival,
whereas N548mu leads to higher cell death in the cultures with respect
to parental ST14A cells.
|
|
Quantitations of these effects by Coulter Counter (Fig.
3a,b) and MTT (which measures
cell viability and/or mitochondrial activity; Fig. 3c,d)
assays confirmed that all clones behaved approximately the same in
normal growth conditions (Fig. 3a,c). However, when
challenged by serum deprivation at 39°C, significant differences in
cell viability and mitochondrial activity were measured (Fig.
3b,d). ST14A cells (open squares) show, in both assays, the typical decrease in cell viability. This effect was more
rapid in N548mu cells (open circles). Remarkably however, when N548wt was overexpressed (open diamonds), cell
viability was maintained even at 72 hr, and decreased only at longer
time points (96 hr). Analysis of protein levels at these later time points at 39°C revealed that the decrease in cell viability
correlated with a reduction in the level of the exogenous protein (data
not shown), further confirming the relationship between expression of
wtHtt and the phenotype observed. This correlation was further substantiated by the finding that subclones that did not express the
exogenous proteins behaved as parental ST14A cells.
View larger version (25K):
[in this window]
[in a new window]
|
Figure 3.
Coulter counter (a, b) and MTT
(c, d) assays on parental ST14A (open
square), N548mu (open circle), and N548wt
(open diamond) cells. a and
c refer to the normal passaging conditions, i.e., 33°C
in serum. b and d indicate the values
obtained after exposure at 39°C in SDM. The graphs show the mean and
SE of MTT assays (n = 12 independent experiments)
and Coulter counter assays (n = 4 independent
experiments) performed on three clones expressing N548wt and on two
clones bearing the N548mu cDNA. Two clones expressing N63wt
(filled diamond) and two expressing FLwt
(asterisk) were analyzed by the same assay. All these
clones behaved as parental ST14A cells in normal passaging
conditions.
|
|
To exclude the possibility that increased cell number in the presence
of N548wt was caused by cell proliferation, a 6 hr BrDU pulse was given
to cells exposed to 39°C for 48 or 72 hr. We found that parental, wt,
and muHtt-expressing cells divided with the same rate at 33°C but, no
division was detected at 39°C in all clones, including N548wt cells
(data not shown). As expected, all clones were also negative for the
Large-T Antigen at 39°C (data not shown). These data indicate that
N548wt acts by increasing cell survival and not cell division.
The pro-survival effect of wtHtt was observed in cells bearing the
N548wt construct. Analogously, expression of FLwt under the same
conditions produced a similar protective effect (Fig. 3d,
asterisk). In contrast, cells expressing a shorter N63 cDNA in the
wt form (Fig. 3d, filled diamond) were not protected and showed the same profile as parental cells. These data indicate that the
protective effect of wtHtt requires a segment of the protein between
aa63 and aa548.
Finally, exposure of the cells to another stress stimulus,
3-nitropropionic acid (3-NP), an inhibitor of complex I activity, revealed a similar protective effect in N548wt and FLwt cells compared
to parental ST14A cells. Figure 4 shows
that cultures of parental ST14A cells exhibited a dose-dependent
decrease in cell viability and/or mitochondrial activity by MTT assay
after 1 and 5 mM 3-NP. This decrease was more evident in
N548mu cells. However, cells from a representative clone expressing
N548wt were almost completely protected by the action of the toxin. The
same was observed in other two N548wt clones (data not shown) and in cells bearing the FLwt protein (Fig. 4). We concluded that wtHtt can
protect from mitochondrial toxins.
View larger version (36K):
[in this window]
[in a new window]
|
Figure 4.
A, Modification in cell viability
and/or mitochondrial activity after 3-NP exposure. Cells were exposed
to the indicated doses of 3-NP and analyzed after 48 hr by MTT assay.
Parental ST14A cells and representative clones expressing N548wt or
N548mu are shown. Protection from 3-NP is also seen in FLwt cells
(*p < 0.05 with respect to parental cells). Shown
is one of three independent experiments performed on the different
clones that gave the same results. B, Hoechst 33258 staining of the supernatant from ST14A cells shows that 3-NP exposure
induces apoptotic cell death.
|
|
MuHtt induces, whereas wtHtt prevents, apoptotic cell death
To determine if the cell death observed in the presence of N548mu
is attributable to apoptosis and if N548wt (and FLwt) prevented DNA
fragmentation, we performed the temperature shift experiment, followed
by DNA analysis or Hoechst staining. As visible in Figure 5A, ST14A cells showed DNA
laddering at 20 hr after the shift. Consistent with the above data, the
appearance of the laddering occurred in N548mu at 1 and 4 hr after the
shift. Remarkably however, cells expressing the N548wt showed no DNA
laddering even at 20 hr after the shift. Similarly, Figure
5B shows that cultures of cells expressing N548mu
(d) present an higher percentage of nuclei with condensed
DNA with respect to parental ST14A cells. This phenomenon was not
observed in N548wt cells (f). These results together indicate that wtHtt directly influences cell survival and acts
as an anti-apoptotic protein in neural cells.
View larger version (50K):
[in this window]
[in a new window]
|
Figure 5.
A, DNA laddering analysis in ST14A
cells, in N548mu cells (8-2 clone), and N548wt cells (7-12 clone)
after the temperature and serum-deprived medium shift. Low molecular
weight DNA samples were prepared 1, 4, and 20 hr after the shift. As a
control, samples from cells in normal growth conditions (33°C) were
included (0 time). These data were replicated in another independent
experiment. B, Hoechst 33258 staining of parental ST14A
(a, b), N548mu (c, d), and N548wt
(e, f) cells at 33°C (a, c, e)
and 39°C (b, d, f). A higher number of cells
with condensed DNA is visible in N548mu cells (d)
with respect to controls (parental ST14A, b). No DNA
condensation is observed in N548wt cells
(f).
|
|
The data reported so far also indicate that N548mu exacerbates
apoptotic cell death in ST14A cells. As discussed later, however, Flmu
cells behave differently from N548mu cells.
WtHtt prevents caspase-3 activation by acting at the level
of caspase-9
In search of the molecular target of wtHtt action, we first
analyzed whether wtHtt affected the activity of the caspase cascade. We
therefore examined the effect of the temperature shift on the activity
of the effector caspase-3. Figure
6A shows caspase-3-like activity measured by monitoring the release of the fluorogenic amc
moiety from the caspase-3-specific substrate Ac-DEVD-amc. As shown,
parental ST14A cells have a low basal level of caspase-3-like activity
in normal growth conditions. Exposure of the cells to SDM at 39°C led
to an increase in activity, in good agreement with the tendency of
ST14A cells to undergo apoptotic cell death in these conditions. Under
the same conditions, a twofold to fivefold greater activity was
measured in N548mu cells. Most significant, the N548wt-expressing cells
had a lower level of caspase-3-like activity than in the parental line
throughout the duration of the experiment (Fig. 6A),
in agreement with the observations presented above. Similar results
were obtained with two other clones expressing N548wt or with the
clones expressing FLwt Htt. These data indicate that wtHtt acts by
inhibiting caspase-3-like activation. To further demonstrate this
point, parental ST14A and cells bearing N548wt or FLwt Htt were
transiently cotransfected with a constitutively active caspase-3
construct and the EGFP plasmid. As shown in Figure 6B, transfection of active caspase-3 kills all cells
efficiently independent of upstream signaling and, according to our
data, after the site where wtHtt exerts its protective effect.
View larger version (20K):
[in this window]
[in a new window]
|
Figure 6.
A, Measurement of caspase-3-like
activity. The release of the fluorogenic amc moiety from the
caspase-3-specific substrate Ac-DEVD-amc is reported at various times
(0, 3, 6, 9, and 12 hr) after the temperature shift. Basal levels of
caspase-3 (time 0, white bar) were obtained at 33°C.
Data are expressed as nanomoles of cleaved substrate. Shown is one of
three experiments performed on different clones that gave similar
results. B, Cotransfection of a constitutively active
caspase-3 together with the EGFP plasmid in parental ST14A (open
bars), N548wtHtt (black bars), and FLwtHtt
(gray bars) cells. Cells were transiently
cotransfected with the active caspase-3 or a control vector (pLXSP)
together with the EGFP-N1 plasmid (Clontech). Cell viability was
determined 30 hr after transfection by counting the ratio of
fluorescent cells (EGFP-positive) versus total number of cells
(*p < 0.01 vs EGFP vector). Data represent the
mean of three independent experiments.
|
|
These data confirm that wtHtt acts upstream of caspase-3 activation.
In an attempt to deliver more defined death stimuli and better identify
the site of action of wtHtt, we next transfected the cells with various
pro-apoptotic genes (Fig. 7). We found
that cells expressing wtHtt were completely protected from death
induced by p55/TNFRI death receptor as well as by the pro-apoptotic
Bcl-2 family members, BIK and BAK. Even after transfection of the
apical caspase-9, the number of TUNEL-positive cells was reduced in
wtHtt-expressing cells.
View larger version (37K):
[in this window]
[in a new window]
|
Figure 7.
TUNEL of the stable cell lines expressing Htt
after transfection with different death inducers. ST14A (dotted
bars), N548wt (7-12 clone, black bars), N548mu
(8-11 clone, horizontal striped bars), and FLmu (10-11
clone, diagonal striped bars) cells were transfected
with 1 µg of the indicated plasmids (0.4 µg for BIK and BAK) or a
control protein. The inset depicts a typical TUNEL
readout. A and B show gating on the
EGFP-expressing cell population. C shows TUNEL staining
of cells transfected with the control protein; D shows
TUNEL staining of cells transfected with p55/TNFR. The control protein
used is 14-3-3. In ST14A cells p55, BIK, BAK, and caspase-9 induce
apoptosis, whereas BAD and caspase-3 show no effect. Apoptosis by all
inducers is blocked in cells expressing wtHtt. FLmu cells show similar
levels of apoptosis as the parental line for p55, BIK, BAK, and
caspase-9 and a marked increase in BAD and caspase-3-induced apoptosis.
This increase is also seen in N548mu cells. Shown is the average of
three independent experiments. Error bars show the average
deviation.
|
|
These data indicate that wtHtt acts upstream of caspase-3 and possibly
at the level of caspase-9 activation.
The caspase-3 zymogen and
BclXL-Bcl2-associated death promoter synergize
with the mutation
Interestingly, whereas transfection of most death inducers had
similar effects in parental ST14A, N548mu, or Flmu cells, transfection of the zymogen (inactive) form of caspase-3 killed the mutant expressing cells efficiently (Fig. 7). The same construct was inefficient in parental or wtHtt-expressing cells (Fig. 7), reflecting the tight control normally exerted on caspase-3 cleavage in these and
other cells (Srinivasula et al., 1998). We concluded that expression of
the poly-Q expanded protein conferred caspase-3 processing ability
selectively in N548mu and Flmu cells. Finally, Figure 7 also shows that
expression of BclXL-Bcl2-associated death promoter (BAD) produced an increase in TUNEL-positive cells only in
clones expressing muHtt constructs. This indicates that muHtt toxicity
feeds into a pathway that requires BAD or a BAD-like function and
argues against a direct activation of caspase-3.
Flmu cells are killed by fewer stimuli compared to
N548mu cells
Of note and surprisingly, whereas FLmu and N548mu cells were both
as efficiently killed by caspase-3 or BAD transfection, FLmu cells were
found to be resistant to other stress stimuli. For example, in contrast
to N548mu cells (Fig. 3), exposure of FLmu cells for various periods of
time to SDM at 39°C did not evoke increased cell demise compared to
parental cells (absorbance ratio at various time points after exposure
to SDM at 39°C with respect to T0 hr, parental
ST14A cells: T2 hr100 ± 7.4; T32
hr82.3 ± 3.3; T48 hr = 48.0 ± 1.8; Flmu cells, 10-6 clone: T2 hr = 100 ± 7.3; T32 hr = 100.3 ± 11.7;
T48 hr = 58.1 ± 6.1; Flmu cells, 10-11
clone: T2 hr = 100 ± 11.2; T32
hr = 84.9 ± 1.9; T48 hr = 57.7 ± 0.8).
Similarly, 3-NP exposure produced a decrease in cell viability and/or
mitochondrial activity in N548mu cells (Fig. 4) but was inefficient in
Flmu cells (absorbance ratio in the presence of 3-NP compared
to untreated cultures, parental ST14A cells: 1 mM = 77.1 ± 7.3; 5 mM = 63.1 ± 4.2; Flmu cells,
10-6 clone: 1 mM = 83.5 ± 6.6; 5 mM = 65.8 ± 4.9; Flmu cells, 10-11 clone: 1 mM = 72.4 ± 7.0; 5 mM = 51.9 ± 2.7). These data
suggest that expression of a CAG as part of the FL protein modifies its
toxicity. Accordingly, cells expressing the full-length protein are
susceptible to fewer death stimuli compared to cells expressing
truncated versions of the mutated protein.
| |
DISCUSSION |
HD is caused by an expanded poly(Q) in Htt, a protein of 340 kDa
molecular weight with cytoplasmic location, and no functional domains
that was found to be crucial during embryonic development. Htt is
widely distributed in the CNS, however it is unlikely that Htt plays a
specific role in neuronal differentiation because Htt/ embryonic
stem cells turn into mature neurons in vitro in the same way
as the wild-type counterparts (Metzler et al., 1999).
To address the question of the exact physiological role of wtHtt in CNS
cells and whether and how it relates to muHtt action, we have obtained
striatal derived cells stably expressing a wt or mu poly(Q) stretch in
the context of the full-length Htt protein or in the N-terminal 548aa
or 63aa truncations. We show that, in this cellular system, wtHtt acts
as a pro-survival molecule. This effect is observed only when
full-length protein or the N548 Htt truncation is expressed.
Further deletion of the protein to an N-terminal 63aa fragment resulted
in the loss of the pro-survival effect. These data indicate that the
protective effect of wtHtt requires at least a segment of the protein
between aa63 and aa548.
Previous work demonstrated that homozygous mice with a targeted
disruption in the Htt gene do not survive to term and suffer early
postimplantation embryonic lethality (Duyao et al., 1995; Nasir et al.,
1995; Zeitlin et al., 1995; White et al., 1997). More recent
morphometric and ultrastructural analysis performed on heterozygous
mice, which survive to adulthood, identified neuronal loss with signs
of apoptosis in the basal ganglia of adult animals (O'Kusky et al.,
1999). In our system, we found that wtHtt specifically interferes with
the cell death machinery. Serum withdrawal and exposure to
mitochondrial toxins led to apoptotic death in parental cells. Mu
Htt-expressing cells were sensitized to these apoptotic stimuli. In
contrast, these stimuli were ineffective in cells expressing wtHtt. The
data indicate that wtHtt directly influences cell survival by acting as
an anti-apoptotic protein. Htt is also an important survival factor
earlier in development because its absence evokes increased apoptosis
in the epiblast, a structure of the embryo known to give rise to the
future ectoderm (Duyao et al., 1995).
To delineate the mechanism of wtHtt anti-apoptotic function, we
transiently expressed several pro-apoptotic genes in the different ST14A subclones. We found that cells expressing wtHtt were protected from cell death induced by death receptors, by the pro-apoptotic Bcl-2
family members (Bik and Bak), as well as by caspase-9. This protection
from multiple apoptotic inducers implies that wtHtt acts on components
of the common death effector pathway. Furthermore, the protection from
pro-apoptotic Bcl-2 homologs implies that wtHtt is acting on
mitochondrial or postmitochondrial apoptotic events. Once caspase-9 is
activated by the apaptosome complex consisting of Apaf-1 and cytochrome
C (Cecconi et al., 1998; Yoshida et al., 1998), it directly activates
the downstream protease, caspase-3 (Zou et al., 1999). The
protection from apoptosis induced by caspase-9 transfection (Fig. 7)
combined with the reduced activity of its downstream effector,
caspase-3, in wtHtt cells (Fig. 6A) and to the
evidence that the same cells could be killed by transfection of a
constitutively active form of caspase-3 (Fig. 6B)
leads us to suggest that wtHtt acts upstream of caspase-3 and likely at the level of caspase-9 activation.
A poly (Q) expansion in the Htt gene is suggested to produce a toxic
gain-of-function effect. Accordingly, expression of muHtt induces cell
death in transgenic animals and in cell culture systems (Cooper et al.,
1998; Hackam et al., 1998; Lunkes and Mandel, 1998; Reddy et al., 1998;
Hodgson et al., 1999). Striatal-derived ST14A cells overexpressing
muHtt undergo a similar fate when challenged with various stress
stimuli (serum withdrawal, 3-NP exposure). In our system we found that,
in the presence of muHtt, DNA fragmentation is accompanied by an
increased caspase-3-like activation, which is consistently above the
level found in parental ST14A cells. These data suggest that the toxic
effect of muHtt converges onto caspase-3. As a further demonstration,
we transfected the zymogen form of caspase-3, which normally does not
lead to apoptosis (Srinivasula et al., 1998), into muHtt expressing
cells and found an increase in the number of TUNEL-positive cells.
Increased activation of caspase-3 and caspase-9 was also recently
reported in lymphoblasts from Huntington's disease patients exposed to
chemical inducers of apoptosis (Sawa et al., 1999). Interestingly,
comparisons of the phenotypes of cells expressing different muHtt
constructs reveal that the toxicity of the polyglutamine repeat is
modulated by the protein backbone. Indeed we found that FLmu protein
promotes cell death only by selective stimuli (BAD or caspase-3
transfections), whereas N548mu truncation promotes death by a larger
range of death stimuli (BAD or caspase-3 transfections, 3-NP
exposure, serum withdrawal). These results highlight the possibility
that truncated versions of poly(Q) proteins induce a wider spectrum of
cytotoxic events than the full-length proteins and support the notion
that proteolytic cleavage is important in HD.
Further analysis of the roles and mechanisms of action of wtHtt will
help to better understand its physiological functions and whether and
how the poly(Q) expansion in the mutated protein interferes with those
and/or other activities.
| |
FOOTNOTES |
Received Oct. 29, 1999; revised March 2, 2000; accepted March 2, 2000.
This work was supported by the Huntington's Disease Society of America
(HDSA), the Hereditary Disease Foundation (HDF), Telethon (Italy
#E840), and Consiglio Nazionale delle Ricerche (Italy, #98.01050.CT04)
to E.C., National Institutes of Health Grants ROI E50811 and DAMD
17-96-1-6085 to C.V., and National Institutes of Health Grant NS16375
to C.A.R. The support of the Istituto Bancario San Paolo di Torino
(Agenzia 8, Milano) to E.C. is also acknowledged. A special thanks to
the members of the HDSA and HDF for their constant and strong
encouragement and to the Associazione Italiana Corea di Huntington
(AICH) for their interest in our work. We thank Drs. Michelle Ehrlich
and Samgram Sisodia for critical discussion and comments on this
manuscript. We also thank Dr. Alnemri E. for providing the active
caspase-3 construct, Mara Monetti for help with cell culture work, and
Ann-Marie DesLaurier for help with the FACS analysis. E.C., M.R.H., and
C.A.R. are members of the "Coalition for the Cure" (HDSA). E.C. is
also a member of the "Cure Initiative" (HDF).
D. R. and J. B. contributed equally to this work.
Correspondence should be addressed to Dr. Elena Cattaneo, Institute of
Pharmacological Sciences, University of Milano, Via Balzaretti 9, 20133 Milano, Italy. E-mail: elena.cattaneo{at}unimi.it.
| |
REFERENCES |
-
Benedetti S,
Pirola B,
Pollo B,
Magrassi L,
Bruzzone MG,
Rigamonti D,
Galli R,
Selleri S,
Di Meco F,
De Fraja C,
Vescovi A,
Cattaneo E,
Finocchiaro G
(2000)
Gene therapy of experimental brain tumors using neural progenitor cells.
Nat Med
4:447-450.
-
Cattaneo E,
Conti L
(1998)
Generation and characterization of embryonic striatal conditionally immortalized ST14A cells.
J Neurosci Res
53:223-234[Web of Science][Medline].
-
Cattaneo E,
Magrassi L,
Santi L,
Butti G,
Giavazzi A,
Pezzotta S
(1994)
A short term analysis of the behaviour of conditionally immortalized progenitors and primary neuroepithelial cells implanted into the fetal rat brain.
Dev Brain Res
83:197-208[Medline].
-
Cattaneo E,
De Fraja C,
Conti L,
Reinach B,
Bolis L,
Govoni S,
Liboi E
(1996a)
Activation of the JAK-STAT pathway leads to proliferation of ST14A CNS progenitor cells.
J Biol Chem
38:23374-23379.
-
Cattaneo E,
Conti L,
Gritti A,
Frolichsthal P,
Govoni S,
Vescovi A
(1996b)
Non-virally mediated gene transfer into human Central Nervous System precursor cells.
Mol Brain Res
42:161-166[Medline].
-
Cecconi F,
Alvarez-Bolado G,
Meyer BI,
Roth KA,
Gruss P
(1998)
Apaf 1 (CED-4 homolog) regulates programmed cell death in mammalian development.
Cell
94:727-737[Web of Science][Medline].
-
Cooper JK,
Schilling G,
Peters MF,
Herring WJ,
Sharp AH,
Kaminsky Z,
Masone J,
Khan FA,
Delanoy M,
Borchelt DR,
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,
Turmaine 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
90:537-548[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].
-
Duyao MP,
Auerbach AB,
Ryan A,
Persichetti F,
Barnes GT,
McNeil SM,
Kowall NW,
Ge P,
Vonsattel JP,
Gusella JF,
Joyner AL,
MacDonald ME
(1995)
Inactivation of the mouse Huntington's disease gene homolog Hdh.
Science
269:407-410[Abstract/Free Full Text].
-
Goldberg YP,
Nicholson DW,
Rasper DM,
Kalchman MA,
Koide HB,
Graham RK,
Bromm M,
Kazemi-Esfajani 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].
-
Gorczyca W,
Gong JP,
Ardelt B,
Traganos F,
Darzynkiewicz Z
(1993)
The cell-cycle related differences in susceptibility of HL-60-cells to apoptosis induced by various antitumor agents.
Cancer Res
53:3186-3192[Abstract/Free Full Text].
-
Gutekunst CA,
Li SH,
Yi H,
Mulroy JS,
Kuemmerle S,
Jones R,
Rye D,
Ferrante RJ,
Hersch SM,
Li XJ
(1999)
Nuclear and neuropil aggregates in Huntington's disease: relationship to neuropathology.
J Neurosci
19:2522-2534[Abstract/Free Full Text].
-
Hackam AS,
Singaraja R,
Wellington CL,
Metzler M,
McCutcheon K,
Zhang T,
Kalchman 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].
-
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
23:1-20[Web of Science][Medline].
-
Huntington's Disease Collaborative Research Group
(1993)
A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosome.
Cell
72:971-983[Web of Science][Medline].
-
Kim M,
Lee HS,
LaForet G,
McIntyre C,
Martin EJ,
Chang P,
Kim TW,
Williams M,
Reddy PH,
Tagle D,
Boyce FM,
Won L,
Heller A,
Aronin N,
DiFiglia M
(1999)
Mutant huntingtin expression in clonal striatal cells: dissociation of inclusion formation and neuronal survival by caspase inhibition.
J Neurosci
19:964-973[Abstract/Free Full Text].
-
Kim TW,
Tanzi RE
(1998)
Neuronal intranuclear inclusions in polyglutamine diseases: nuclear weapons or nuclear fallout?
Neuron
21:657-659[Web of Science][Medline].
-
Kowall NW,
Ferrante RJ,
Martin JB
(1987)
Pattern of cell loss in Huntington's disease.
Trends Neurosci
10:24-29.
-
Lundberg C,
Martinez-Serrano A,
Cattaneo E,
McKay RDG,
Bjorklund A
(1997)
Survival, integration and differentiation of neural stem cell lines after transplantation to the adult rat striatum.
Exp Neurol
145:342-360[Web of Science][Medline].
-
Lunkes A,
Mandel JL
(1998)
A cellular model that recapitulates major pathogenic steps of Huntington's disease.
Hum Mol Genet
7:1355-1361[Abstract/Free Full Text].
-
MacDonald ME,
Gusella J
(1996)
Huntington's disease: translating a CAG repeat into a pathogenic mechanism.
Curr Opin Neurobiol
6:638-643[Web of Science][Medline].
-
Magrassi L,
Ehrlich ME,
Butti G,
Pezzotta S,
Govoni S,
Cattaneo E
(1998)
Basal ganglia precursors found in aggregates following embryonic transplantation adopt a striatal phenotype in heterotopic locations.
Development
125:2847-2855[Abstract].
-
Metzler M,
Chen N,
Helgason CD,
Graham RK,
Nichol K,
McCutcheon K,
Nasir J,
Humphries RK,
Raymond LA,
Hayden MR
(1999)
Life without huntingtin: normal differentiation into functional neurons.
J Neurochem
72:1009-1018[Web of Science][Medline].
-
Nasir J,
Floresco SB,
O'Kusky JR,
Diewert VM,
Richman J,
Zeisler J,
Borowski A,
Marth JD,
Phillips AG,
Hayden MR
(1995)
Targeted disruption of the Huntington's disease gene results in embryonic lethality and behavioural and morphological changes in heterozygotes.
Cell
81:811-823[Web of Science][Medline].
-
Ona VO,
Li M,
Vonsattel JP,
Andrews LJ,
Khan SQ,
Chung WM,
Frey AS,
Menon AS,
Li XJ,
Stieg PE,
Yuan J,
Penney JB,
Young AB,
Cha JH,
Friedlander RM
(1999)
Inhibition of caspase-1 slows disease progression in a mouse model of Huntington's disease.
Nature
399:263-267[Medline].
-
O'Kusky JR,
Nasir J,
Cicchetti F,
Parent A,
Hayden MR
(1999)
Neuronal degeneration in the basal ganglia and loss of pallido-subthalamic synapses in mice with targeted disruption of the Huntington's disease gene.
Brain Res
818:468-479[Web of Science][Medline].
-
Reddy PH,
Williams M,
Charles V,
Garrett L,
Pike-Buchanan L,
Whetsell Jr WO,
Miller G,
Tagle DA
(1998)
Behavioral abnormalities and selective neuronal loss in HD transgenic mice expressing mutated full-length HD cDNA.
Nat Genet
20:198-202[Web of Science][Medline].
-
Reddy PH,
Williams M,
Tagle DA
(1999)
Recent advances in understanding the pathogenesis of Huntington's disease.
Trends Neurosci
22:248-255[Web of Science][Medline].
-
Ross CA,
Margolis RL,
Rosenblatt A,
Ranen NG,
Becher MW,
Aylward EA
(1997)
Reviews in molecular medicine: Huntington's disease and a related disorder, dentatorubral-pallidoluysian atrophy (DRPLA).
Medicine
76:305-338[Medline].
-
Sanchez I,
Xu CJ,
Juo P,
Kakizaka A,
Blenis J,
Yuan J
(1999)
Caspase-8 is required for cell death induced by expanded polyglutamine repeats.
Neuron
22:623-633[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].
-
Sawa A,
Wiegand GW,
Cooper J,
Margolis LM,
Sharp AH,
Lawler Jr JF,
Greenamyre T,
Snyder SH,
Ross CA
(1999)
Increased apoptosis of Huntington disease lymphoblasts associated with repeat length-dependent mitochondrial depolarization.
Nat Med
5:1194-1198[Web of Science][Medline].
-
Sharp AH,
Ross CA
(1996)
Neurobiology of Huntington's disease.
Neurobiol Dis
3:3-15[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].
-
Srinivasula SM,
Ahmad M,
MacFarlane M,
Luo Z,
Fernandes-Alnemri T,
Alnemri ES
(1998)
Generation of constitutively active recombinant caspases-3 and -6 by rearrangement of their subunits.
J Biol Chem
273:10107-10111[Abstract/Free Full Text].
-
Vonsattel JP,
Myers RH,
Stevens TJ,
Ferrante RJ,
Bird ED,
Richardson EP
(1985)
Neuropathological classification of Huntington's disease.
J Neuropathol Exp Neurol
44:559-577[Web of Science][Medline].
-
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].
-
White JK,
Auerbach W,
Duyao MP,
Vonsattel JP,
Gusella JF,
Joyner AL,
MacDonald ME
(1997)
Huntingtin is required for neurogenesis and is not impaired by Huntington's disease CAG expansion.
Nat Genet
17:404-410[Web of Science][Medline].
-
Yoshida H,
Kong YY,
Yoshida R,
Elia AJ,
Hakem A,
Hakem R,
Penninger JM,
Mak TW
(1998)
Apaf 1 is required for mitochondrial pathways of apoptosis and brain development.
Cell
94:739-750[Web of Science][Medline].
-
Zeitlin S,
Liu JP,
Chapman DL,
Papaioannou VE,
Esfratiadis A
(1995)
Increased apoptosis and early embryonic lethality in mice nullizygous for the Huntington's disease gene homologue.
Nat Genet
11:155-162[Web of Science][Medline].
-
Zou H,
Li Y,
Liu X,
Wang X
(1999)
An APAF-1 cytochrome c multimeric complex is a functional apaptosome that activates procaspase-9.
J Biol Chem
274:11549-11556[Abstract/Free Full Text].
Copyright © 2000 Society for Neuroscience 0270-6474/00/20103705-09$05.00/0
This article has been cited by other articles:
|
|
|
|
 
N. A. Aziz, C. K. Jurgens, G. B. Landwehrmeyer, EHDN Registry Study Group, W.M.C. van Roon-Mom, G. J.B. van Ommen, T. Stijnen, and R. A.C. Roos
Normal and mutant HTT interact to affect clinical severity and progression in Huntington disease
Neurology,
October 20, 2009;
73(16):
1280 - 1285.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Zhang, M. B. Feany, S. Saraswati, J. T. Littleton, and N. Perrimon
Inactivation of Drosophila Huntingtin affects long-term adult functioning and the pathogenesis of a Huntington's disease model
Dis. Model. Mech.,
May 1, 2009;
2(5-6):
247 - 266.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Ermak, K. J. Hench, K. T. Chang, S. Sachdev, and K. J. A. Davies
Regulator of Calcineurin (RCAN1-1L) Is Deficient in Huntington Disease and Protective against Mutant Huntingtin Toxicity in Vitro
J. Biol. Chem.,
May 1, 2009;
284(18):
11845 - 11853.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Luo and D. C. Rubinsztein
Huntingtin promotes cell survival by preventing Pak2 cleavage
J. Cell Sci.,
March 15, 2009;
122(6):
875 - 885.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L.-S. Her and L. S. B. Goldstein
Enhanced Sensitivity of Striatal Neurons to Axonal Transport Defects Induced by Mutant Huntingtin
J. Neurosci.,
December 10, 2008;
28(50):
13662 - 13672.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Wang, S. Zhu, Z. Pei, M. Drozda, I. G. Stavrovskaya, S. J. Del Signore, K. Cormier, E. M. Shimony, H. Wang, R. J. Ferrante, et al.
Inhibitors of Cytochrome c Release with Therapeutic Potential for Huntington's Disease
J. Neurosci.,
September 17, 2008;
28(38):
9473 - 9485.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Benchoua, Y. Trioulier, E. Diguet, C. Malgorn, M.-C. Gaillard, N. Dufour, J.-M. Elalouf, S. Krajewski, P. Hantraye, N. Deglon, et al.
Dopamine determines the vulnerability of striatal neurons to the N-terminal fragment of mutant huntingtin through the regulation of mitochondrial complex II
Hum. Mol. Genet.,
May 15, 2008;
17(10):
1446 - 1456.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Tartari, C. Gissi, V. Lo Sardo, C. Zuccato, E. Picardi, G. Pesole, and E. Cattaneo
Phylogenetic Comparison of Huntingtin Homologues Reveals the Appearance of a Primitive polyQ in Sea Urchin
Mol. Biol. Evol.,
February 1, 2008;
25(2):
330 - 338.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Varma, R. Cheng, C. Voisine, A. C. Hart, and B. R. Stockwell
Inhibitors of metabolism rescue cell death in Huntington's disease models
PNAS,
September 4, 2007;
104(36):
14525 - 14530.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Nopoulos, V. A. Magnotta, A. Mikos, H. Paulson, N. C. Andreasen, and J. S. Paulsen
Morphology of the Cerebral Cortex in Preclinical Huntington's Disease
Am J Psychiatry,
September 1, 2007;
164(9):
1428 - 1434.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Rigamonti, D. Bolognini, C. Mutti, C. Zuccato, M. Tartari, F. Sola, M. Valenza, A. G. Kazantsev, and E. Cattaneo
Loss of Huntingtin Function Complemented by Small Molecules Acting as Repressor Element 1/Neuron Restrictive Silencer Element Silencer Modulators
J. Biol. Chem.,
August 24, 2007;
282(34):
24554 - 24562.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. L. Anne, F. Saudou, and S. Humbert
Phosphorylation of Huntingtin by Cyclin-Dependent Kinase 5 Is Induced by DNA Damage and Regulates Wild-Type and Mutant Huntingtin Toxicity in Neurons
J. Neurosci.,
July 4, 2007;
27(27):
7318 - 7328.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Coufal, M. M. Maxwell, D. E. Russel, A. M. Amore, S. M. Altmann, Z. R. Hollingsworth, A. B. Young, D. E. Housman, and A. G. Kazantsev
Discovery of a Novel Small-Molecule Targeting Selective Clearance of Mutant Huntingtin Fragments
J Biomol Screen,
April 1, 2007;
12(3):
351 - 360.
[Abstract]
[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. M. Van Raamsdonk, W. T. Gibson, J. Pearson, Z. Murphy, G. Lu, B. R. Leavitt, and M. R. Hayden
Body weight is modulated by levels of full-length Huntingtin
Hum. Mol. Genet.,
May 1, 2006;
15(9):
1513 - 1523.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Pal, F. Severin, B. Lommer, A. Shevchenko, and M. Zerial
Huntingtin-HAP40 complex is a novel Rab5 effector that regulates early endosome motility and is up-regulated in Huntington's disease.
J. Cell Biol.,
February 13, 2006;
172(4):
605 - 618.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Valenza, D. Rigamonti, D. Goffredo, C. Zuccato, S. Fenu, L. Jamot, A. Strand, A. Tarditi, B. Woodman, M. Racchi, et al.
Dysfunction of the Cholesterol Biosynthetic Pathway in Huntington's Disease
J. Neurosci.,
October 26, 2005;
25(43):
9932 - 9939.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. C. Warby, E. Y. Chan, M. Metzler, L. Gan, R. R. Singaraja, S. F. Crocker, H. A. Robertson, and M. R. Hayden
Huntingtin phosphorylation on serine 421 is significantly reduced in the striatum and by polyglutamine expansion in vivo
Hum. Mol. Genet.,
June 1, 2005;
14(11):
1569 - 1577.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. M. Van Raamsdonk, J. Pearson, D. A. Rogers, N. Bissada, A. W. Vogl, M. R. Hayden, and B. R. Leavitt
Loss of wild-type huntingtin influences motor dysfunction and survival in the YAC128 mouse model of Huntington disease
Hum. Mol. Genet.,
May 15, 2005;
14(10):
1379 - 1392.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Bjorkqvist, M. Fex, E. Renstrom, N. Wierup, A. Petersen, J. Gil, K. Bacos, N. Popovic, J.-Y. Li, F. Sundler, et al.
The R6/2 transgenic mouse model of Huntington's disease develops diabetes due to deficient {beta}-cell mass and exocytosis
Hum. Mol. Genet.,
March 1, 2005;
14(5):
565 - 574.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Gambarotta, D. Garzotto, E. Destro, B. Mautino, C. Giampietro, S. Cutrupi, C. Dati, E. Cattaneo, A. Fasolo, and I. Perroteau
ErbB4 Expression in Neural Progenitor Cells (ST14A) Is Necessary to Mediate Neuregulin-1{beta}1-induced Migration
J. Biol. Chem.,
November 19, 2004;
279(47):
48808 - 48816.
[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]
|
|
|
|
|
|
|
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]
|
|
|
|
|
|
|
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]
|
|
|
|
|
|
|
X. Wang, S. Zhu, M. Drozda, W. Zhang, I. G. Stavrovskaya, E. Cattaneo, R. J. Ferrante, B. S. Kristal, and R. M. Friedlander
Minocycline inhibits caspase-independent and -dependent mitochondrial cell death pathways in models of Huntington's disease
PNAS,
September 2, 2003;
100(18):
10483 - 10487.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Cattaneo
Dysfunction of Wild-Type Huntingtin in Huntington disease
Physiology,
February 1, 2003;
18(1):
34 - 37.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. W Scott, C. Sobotka-Briner, D. E. Wilkins, R. T. Jacobs, J. J. Folmer, W. J. Frazee, R. V. Bhat, S. V. Ghanekar, and D. Aharony
Novel Small Molecule Inhibitors of Caspase-3 Block Cellular and Biochemical Features of Apoptosis
J. Pharmacol. Exp. Ther.,
January 1, 2003;
304(1):
433 - 440.
[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]
|
|
|
|
|
|
|
C. L. Wellington, L. M. Ellerby, C.-A. Gutekunst, D. Rogers, S. Warby, R. K. Graham, O. Loubser, J. van Raamsdonk, R. Singaraja, Y.-Z. Yang, et al.
Caspase Cleavage of Mutant Huntingtin Precedes Neurodegeneration in Huntington's Disease
J. Neurosci.,
September 15, 2002;
22(18):
7862 - 7872.
[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]
|
|
|
|
|
|
|
S. Sipione, D. Rigamonti, M. Valenza, C. Zuccato, L. Conti, J. Pritchard, C. Kooperberg, J. M. Olson, and E. Cattaneo
Early transcriptional profiles in huntingtin-inducible striatal cells by microarray analyses
Hum. Mol. Genet.,
August 15, 2002;
11(17):
1953 - 1965.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. P. Lieberman, G. Harmison, A. D. Strand, J. M. Olson, and K. H. Fischbeck
Altered transcriptional regulation in cells expressing the expanded polyglutamine androgen receptor
Hum. Mol. Genet.,
August 15, 2002;
11(17):
1967 - 1976.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. P. de Almeida, C. A. Ross, D. Zala, P. Aebischer, and N. Deglon
Lentiviral-Mediated Delivery of Mutant Huntingtin in the Striatum of Rats Induces a Selective Neuropathology Modulated by Polyglutamine Repeat Size, Huntingtin Expression Levels, and Protein Length
J. Neurosci.,
May 1, 2002;
22(9):
3473 - 3483.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Zhou, S.-H. Li, and X.-J. Li
Chaperone Suppression of Cellular Toxicity of Huntingtin Is Independent of Polyglutamine Aggregation
J. Biol. Chem.,
December 14, 2001;
276(51):
48417 - 48424.
[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]
|
|
|
|
|
|
|
A. Reiner, N. Del Mar, C. A. Meade, H. Yang, I. Dragatsis, S. Zeitlin, and D. Goldowitz
Neurons Lacking Huntingtin Differentially Colonize Brain and Survive in Chimeric Mice
J. Neurosci.,
October 1, 2001;
21(19):
7608 - 7619.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. W Ho, R. Brown, M. Maxwell, A. Wyttenbach, and D. C Rubinsztein
Wild type huntingtin reduces the cellular toxicity of mutant huntingtin in mammalian cell models of Huntington's disease
J. Med. Genet.,
July 1, 2001;
38(7):
450 - 452.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-M. Lecerf, T. L. Shirley, Q. Zhu, A. Kazantsev, P. Amersdorfer, D. E. Housman, A. Messer, and J. S. Huston
Human single-chain Fv intrabodies counteract in situ huntingtin aggregation in cellular models of Huntington's disease
PNAS,
April 10, 2001;
98(8):
4764 - 4769.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Trettel, D. Rigamonti, P. Hilditch-Maguire, V. C. Wheeler, A. H. Sharp, F. Persichetti, E. Cattaneo, and M. E. MacDonald
Dominant phenotypes produced by the HD mutation in STHdhQ111 striatal cells
Hum. Mol. Genet.,
November 1, 2000;
9(19):
2799 - 2809.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Disease Mechanisms in Neuroscience
Neuroscientist,
October 1, 2000;
6(5):
304 - 304.
[PDF]
|
|
|
|
|
|
|
C. Zuccato, A. Ciammola, D. Rigamonti, B. R. Leavitt, D. Goffredo, L. Conti, M. E. MacDonald, R. M. Friedlander, V. Silani, M. R. Hayden, et al.
Loss of Huntingtin-Mediated BDNF Gene Transcription in Huntington's Disease
Science,
July 20, 2001;
293(5529):
493 - 498.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Rigamonti, S. Sipione, D. Goffredo, C. Zuccato, E. Fossale, and E. Cattaneo
Huntingtin's Neuroprotective Activity Occurs via Inhibition of Procaspase-9 Processing
J. Biol. Chem.,
April 27, 2001;
276(18):
14545 - 14548.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. F. Peters and C. A. Ross
Isolation of a 40-kDa Huntingtin-associated Protein
J. Biol. Chem.,
January 26, 2001;
276(5):
3188 - 3194.
[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]
|
|
|
|
|
|
|
X. Wang, J. H. Bauer, Y. Li, Z. Shao, F. S. Zetoune, E. Cattaneo, and C. Vincenz
Characterization of a p75NTR Apoptotic Signaling Pathway Using a Novel Cellular Model
J. Biol. Chem.,
August 31, 2001;
276(36):
33812 - 33820.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
