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 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
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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
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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.
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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.
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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.
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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.
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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).
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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.
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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.
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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.
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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.
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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.
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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.
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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).
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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.
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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.
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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.
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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.
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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.
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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.
| |
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TOP
ABSTRACT
INTRODUCTION
