From the Department of Pharmacological Sciences, University of Milano, Via Balzaretti 9, 20133 Milano, Italy
Received for publication, January 26, 2001, and in revised form, March 2, 2001
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ABSTRACT |
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Huntington's Disease is an inherited
neurodegenerative disease that affects the medium spiny neurons in the
striatum. The disease is caused by the expansion of a polyglutamine
sequence in the N terminus of Huntingtin (Htt), a widely expressed
protein. Recently, we have found that Htt is an antiapoptotic protein
in striatal cells and acts by preventing caspase-3 activity. Here we
report that Htt overexpression in other CNS-derived cells can protect
them from more than 20 days exposure to fatal stimuli. In particular,
we found that cytochrome c continues to be released from
mitochondria into the cytosol of cells that overexpress normal Htt.
However, procaspase-9 is not processed, indicating that wild-type Htt
(wtHtt) acts downstream of cytochrome c release.
These data show that Htt inhibits neuronal cell death by
interfering with the activity of the apoptosome complex.
Huntingtin is a protein that is enriched in neurons and exerts a
function in the control of neuronal survival and stability (1).
Interest in this protein derives from the fact that an expanded
polyglutamine (poly(Gln)) tract in its N terminus causes Huntington's
Disease (HD),1 a neurological
disorder associated with the selective loss of striatal and cortical
neurons (2). It is well documented that the extended poly(Gln) stretch
in Htt confers a deleterious gain-of-function to the protein. More
recently, the hypothesis that loss of normal Htt function might
contribute to the disease has gained strong support from studies
reporting a number of beneficial functions of this protein in CNS cells
(Refs. 3-5; reviewed in Ref. 1). In addition, Ona et al.
(6) demonstrate that in HD transgenic mice there is a depletion of
endogenous Htt.
Early studies indicate that Htt serves important functions during
normal embryonic development, because homozygous knockout mice die by
day 7.5 (7-9). On the other hand, recent findings obtained in adult
mice where conditional inactivation of wild-type Htt was elicited in
mature neurons demonstrate degeneration of axon fibers and evident
signs of apoptosis, which were accompanied by neurological dysfunctions
similar to those observed in current transgenic mouse models of HD (5).
More direct evidence of Htt function in cell survival was provided for
the first time by a study from our group, where conditionally
immortalized striatal derived ST14A cells engineered to express the
normal or mutant protein were exposed to various apoptotic stimuli such
as serum withdrawal, 3-nitroproprionic acid, or transfection of death
genes (4). We found that these stimuli triggered apoptotic death in
parental ST14A cells but were ineffective in ST14A derivatives expressing either the FL or an N terminus 548-amino acid truncation of
the wild-type Htt (FLwt and N548wt, respectively). As expected, an
increased propensity for death was observed in subclones expressing mutant Htt. A subsequent report suggests that the Htt antiapoptotic effect occurs via sequestration of the HIP1 proapoptotic molecule (10).
Other investigations imply a role for Htt in protein-protein interactions, modulation of gene transcription as well as in retrograde and fast axonal transport, and nuclear-cytoplasmic shuttling (for reviews, see Refs. 1 and 11). Taken together, these data indicate that
Htt exerts important effects on neuronal cell survival and stability.
We have also reported that wtHtt cells are protected from apoptosis
induced by caspase-9 transfection (4). These data, combined with the
reduced activity of caspase-3 and evidence that the same cells are
killed by transfection of a constitutively active form of caspase-3,
suggest strongly that wtHtt acts upstream of caspase-3, probably at the
level of caspase-9 (4).
Here we show that Htt's neuroprotective activity does not occur via
blockade of cytochrome c release from the mitochondria, but
rather through inhibition of cytochrome
c-dependent procaspase-9 processing and
activity. One hypothesis is that Htt modulates the activity of the
apoptosome complex by interacting with one of the apoptosome
components. Finally, we report that Htt permanently protects CNS cells
from toxic insults and that this effect occurs in several different
CNS-derived cells.
Cell Culture and MTT Assay--
Conditionally immortalized
striatal-derived ST14A cells and subclones previously engineered to
overexpress the full-length wild-type (FLwt) or full-length mutant
(FLmu) proteins or an amino terminus 548-amino acid portion of
Huntingtin (N548) in the wild-type or mutant versions (N548wt or N548mu
cells, respectively) were utilized in this study and grown as described
previously (4). The different clones express the exogenous normal or
mutant Huntingtin at a similar level (4). Hippocampal-derived HiB5
cells (12) were grown at 33 °C in the presence of Dulbecco's
modified Eagle's medium (4). L929 cells and derivatives were grown at
37 °C in complete medium. Chemically defined serum-free medium (SDM) was prepared as described (4). Infection was performed using medium
conditioned from RetroPack PT67 Cell Line
(CLONTECH) stably transfected with the cDNA of
interest and according to previously described procedures (13). The MTT
assay was performed as described previously (4).
Measurement of Caspase-9 and Caspase-8
Activities--
Samples of 1-2 × 106 cells were
rinsed in cold phosphate-buffered saline, lysed, and stored as
described (4). Lysates were then incubated at 37 °C in buffer
supplemented with substrate LEHD-7-amino-4-trifluoromethyl
coumarin (afc) (25 µM) for caspase-9 or IETD-afc (25 µM) for caspase-8. Release of the fluorogenic afc moiety
and protein contents were quantified as described previously (4). To
assess specificity, caspase-9 and caspase-8 inhibitors were utilized
(Z-LEHD-FMK and Z-LETD-FMK, respectively). Where indicated, 5 nM cytochrome c was added.
Cell Fractionation--
ST14A cells and subclones overexpressing
FLwt or FLmu were plated onto 100-mm dishes. At 80% confluence,
cells were exposed to SDM for 4 h at 39 °C, and the cytosolic
and mitochondrial fractions were prepared according to a protocol
kindly provided by Dr. T. Greenamyre and Dr. A. Panov. Briefly, cells
were lysed in a hypotonic buffer and homogenized, and tonicity was
adjusted to 250 mosM. Subsequent centrifugations at
9,300 × g pelleted the mitochondrial fraction. The
supernatant was centrifuged at 100,000 × g to obtain the cytosolic fraction.
Western Blot Analyses--
Total cellular lysates were obtained
with a hypotonic buffer containing Triton (0.5%). The blotted proteins
were exposed to anti-Htt mAb2166 (dilution 1:5000, Chemicon) or
anti-cytochrome c (dilution 1:2000, PharMingen) antibodies.
Procaspase-9 Processing--
Five 150-mm dishes of cells
at 90% confluence were lysed with Buffer A. The lysates were
centrifuged at 100,000 × g to obtain the S-100
fraction. 35S-Labeled procaspase-9 was prepared using the
TNT-quick in vitro-transcription/translation kit (Promega).
In the assay, 20 µg of S-100 extracts were incubated with
35S-procaspase-9, 1 mM dATP, and increasing
amounts of cytochrome c at 37 °C for 30 min.
Reactions were run on 12% SDS-PAGE, and the dried gel was
autoradiographed. Statistical analyses were performed by analysis of
variance test as described in Rigamonti et al. (4).
Permanent Inhibition of Cell Death by Htt Expression in Various CNS
Cell Types--
ST14A cells (14), which are immortalized with the
temperature sensitive oncogene Large T-Antigen, stop dividing at the
non-permissive temperature (39 °C), tend to differentiate, and, in
chemically defined SDM undergo apoptotic cell death. ST14A subclones
stably transfected with wtHtt cDNA are protected from this death
stimulus (4). Yet, protection by Htt was drastically reduced after
120 h of exposure to this condition as a consequence of the
disappearance of the exogenous protein (4). Therefore, the possibility
of a permanent, long term protective effect of Htt in these engineered striatal cells could not be properly assessed.
We have now obtained a new generation of subclones of striatal ST14A
cells where the expression of the transgene remains constant with time
of exposure at the non-permissive temperature. N548wt cDNA was
retrovirally transduced into ST14A cells because it reproduces fully
the effect of the FL protein. Fig.
1A shows a Western blot analysis of lysates obtained from wtHtt cells that were exposed to
39 °C in SDM for 0, 5, 15, and 20 days. As shown, N548wt Htt expression remained stable over time.
In these new ST14A subclones (Fig. 1B, iN548wt-32 (
To determine whether the antiapoptotic effect of Htt was specific for
the striatal-derived cells, the N548wt cDNA was also expressed in
hippocampal cells HiB5, fibroblast 3T3 cells, and fibrosarcoma L929
cells. Whereas overexpression of Htt in HiB5 cells confirmed the
protective effect from SDM (Fig. 1D) or 3-nitropropionic acid exposures (Fig. 1E), no protection was seen in 3T3
derivatives with stable expression of wtHtt treated with
3-nitropropionic acid (data not shown) or in N548wt-expressing
subclones of L929 cells exposed to tumor necrosis factor (TNF) (Fig.
1F). In these same conditions, no changes in caspase-8
activity were detected in N548wt L929 cells with respect to parental
cells (data not shown).
Decreased Basal and Cytochrome c-stimulated Caspase-9 Activity in
FLwt Cells--
We have previously shown that wtHtt protects from
apoptosis upstream of caspase-3 and downstream of proapoptotic Bcl-2
family members BIK and BAK (4). Also, the toxicity that follows
transfection of procaspase-9 in parental ST14A cells is prevented by
expression of wtHtt (4). On this basis, we hypothesized that the
molecular target of Htt neuroprotective action lies at the level of
caspase-9.
We therefore analyzed the effect of the temperature shift on the
activity of caspase-9 by monitoring the release of the fluorogenic afc
moiety from the caspase-9 substrate LEHD-afc in the absence or presence
of exogenous cytochrome c. In Fig.
2A, parental ST14A cells (
We concluded that wild-type Htt exerts its antiapoptotic effect by
modulating a step upstream of caspase-9 activation.
Huntingtin's Antiapoptotic Effect Occurs Downstream of
Mitochondrial Cytochrome c Release--
We previously reported that
wtHtt cells are protected from the action of proapoptotic Bcl-2
homologs, implying that Htt acts on mitochondrial or postmitochondrial
apoptotic events (4). To evaluate whether inhibition of caspase-9
activity in wtHtt cells is caused by impaired cytochrome c
release from the mitochondria, we analyzed levels of cytosolic
cytochrome c in parental, FLwt, and FLmu cells. Cells were
exposed to SDM at 39 °C, and the cytosolic and mitochondrial
fractions were prepared. As shown in Fig.
3, under normal growth conditions most of
the cytochrome c is found in the mitochondrial fraction.
Indeed, a Western analysis of the cytosolic fraction from parental and
FLwt cells was negative for cytochrome c. Interestingly, in
the same normal passaging conditions, cytochrome c was found
in the cytosol of FLmu cells. Notably, however, exposure of the cells
to a death stimulus like SDM at 39 °C for 4 h evoked a similar
release of cytochrome c from the mitochondria of parental
and FLwt cells (Fig. 3). We concluded that Htt protective activity does
not involve inhibition of cytochrome c release from the
mitochondria.
WtHtt Inhibits Cleavage of Procaspase-9--
Release of cytochrome
c from mitochondria by apoptotic signals induces
ATP/dATP-dependent formation of the apoptosome complex, with subsequent autoprocessing of procaspase-9 into its two active fragments (15). Because our data demonstrate inhibition of caspase-9 activity in the presence of cytochrome c in the cytosolic
fraction of wtHtt cells, we reasoned that Htt could play a role in
caspase-9 processing. To this end, in vitro caspase-9
cleavage assays were performed (16). S-100 extracts from parental ST14A
cells, N548wt, FLwt, N548mu, and Flmu cells were incubated with
35S-labeled in vitro transcribed/translated
procaspase-9, and increasing amounts (0.4, 4, 40 ng/µl) of cytochrome
c. Fig. 4 shows that the
appearance of the two active fragments from the inactive 48-kDa procaspase-9 in parental ST14A cells is cytochrome c
concentration-dependent. In particular, no processing is
observed at the lower concentration of 0.4 ng/µl. In contrast, at
that same dose, N548mu and FLmu cells were able to process
procaspase-9. Thus, we also demonstrate that N548wt and FLwt cells
process procaspase-9 less efficiently than parental cells, even at the
maximal concentration of cytochrome c utilized in our study.
We conclude that wtHtt exerts its antiapoptotic role by interfering
with cytochrome c-dependent procaspase-9
processing.
In our previous report we demonstrated that Htt prevents apoptosis
of striatal cells (4). This evidence, combined with more recent data
from other authors, together suggest that loss of Htt function plays a
role in HD (1).
The mechanisms by which Htt exerts this protective effect is also
beginning to be elucidated. We previously found that Htt acts upstream
of caspase-3 and downstream of proapoptotic Bcl2-members (4). Here we
report an inhibition of caspase-9 activity in cells expressing wtHtt.
We also report that cytochrome c redistributes normally into
the cytosol of wtHtt cells exposed to apoptotic insults. However, these
cells do not undergo cell death (4). During apoptosis cytochrome
c release from mitochondria yields to recruitment of Apaf-1
and processing of procaspase-9 into its two active fragments. We
demonstrate that wild-type Htt exerts a negative effect on cytochrome
c-dependent processing of procaspase-9. As expected,
processing of procaspase-9 occurs more efficiently in mutant Htt cells,
leading to increased caspase-3 activation; the latter result was
reported in several studies (4, 17). We also show that cells expressing
the mutation have a basal leakage of cytochrome c, which may
reflect an acquired activity of mutant Htt at a mitochondrial or
premitochondrial level. These data are in agreement with data showing
caspase-1 and caspase-8 involvement in HD (6, 18) and with the
demonstration of cytochrome c leakage in PC12 cells
expressing the exon 1 portion of mutant Htt (19).
Based on the described activities of Htt, we also hypothesize that the
wild-type protein functions to counteract caspase-9 activation either
by binding to it directly or via inhibition of the apoptosome complex
formation. In particular, Htt appears specifically to inhibit an
apoptotic pathway, which is likely the most active death pathway in CNS
cells. In fact, we found that Htt does not interfere with another
apoptotic death pathway, which is caspase-8 mediated. These data
reinforce the hypothesis that Htt plays a key role in CNS cells. As a
matter of fact, we found that Htt is equally neuroprotective in a
number of different CNS-derived cells. We also found that cells
expressing exogenous Htt do not die even after lengthy exposure to
apoptotic insults. Such an effect is of potential therapeutical
value, as strategies aimed at restoring or increasing normal Htt levels
or activities in cells would be relevant for HD and other
neurodegenerative diseases.
Taken together, these data imply that Htt plays an important role in
the formation and/or function of the apoptosome complex, the controlled
activity of which is crucial for proper CNS cell survival. At the same
time, our findings argue strongly in favor of the possibility that loss
of neuroprotective Htt function occurs in HD, with resulting increased
vulnerability of CNS neurons (reviewed in Ref. 1).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (27K):
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Fig. 1.
Huntingtin protects CNS cells from lengthy
exposure to apoptotic stimuli. A, Western blot analyses
showing permanent expression of N548wt Htt in the newly generated ST14A
subclones exposed for 0, 5, 15, and 20 days to apoptotic insults (SDM
at 39 °C). B, MTT assay, ST14A cells ( ), and two
N548wt infected subclones, iN548-32 (
) and iN548wt-36 (
), were
exposed to the same conditions as in A. Cell survival was
monitored at the times indicated. Parental ST14A cells gradually
underwent apoptotic cell death. By contrast, cells overexpressing
wild-type Htt survived over 20 days of exposure to this condition. The
graph shows the mean ± S.D. of four independent
assays. The same data were obtained when cell viability was measured
using a Coulter Counter machine (not shown; Ref. 4). C, MTT
assay, 3-nitropropionic acid was added for 48 h to wtHtt cells
after a 20-day exposure under conditions described in A.
Protection from death induced by this toxin was observed in both clones
expressing wtHtt. Treatment of parental cells with 3-nitropropionic
acid normally leads to cell death. Indeed, in a parallel experiment,
3-nitropropionic acid evoked dose-dependent death of
parental ST14A cells in normal growth conditions (not shown; Ref. 4).
D, hippocampal-derived HiB5 cells were retrovirally
transduced with N548wt and exposed to the same experimental paradigm as
in A. Expression of the transgene was confirmed by Western
blot (not shown). In the graph, wtHtt-HiB5 cells (
) were
protected from death with respect to parental HiB5 cells (
).
E, improved viability of wtHtt-HiB5 cells following
3-nitropropionic acid exposure. MTT assay, 48-h time point; *,
p < 0.05 and **, p < 0.001 with
respect to parental cells. F, L929 cells overexpressing
N548wt (wtHtt-L929 cells) were not protected from a 24-h exposure to
different doses of TNF. Inset, Western blot showing N548wt
protein expression in one of the engineered L929 subclones (lane
2), and no immunoreactivity in parental cells (lane
1).
) and
iN548wt-36 (
), permanent protection from the apoptotic insult occurs even after a 20-day exposure to these conditions. In the figure, parental ST14A cells show the typical decrease in cell survival with
time at 39 °C (
). Furthermore, application of 3-nitropropionic acid at that time point (20 days at 39 °C) did not evoke death in
iN548wt-32 and iN548wt-36 cells (Fig. 1C), confirming a
permanent antiapoptotic effect of the exogenous wtHtt. In a control
experiment, 3-nitropropionic acid evoked dose-dependent
cell death in parental ST14A cells grown in normal passaging conditions
(data not shown; Ref. 4).
)
show an increased release of the fluorogenic moiety over time, which,
as expected, is enhanced in FLmu cells (
). In contrast, release of
the fluorogenic moiety is completely inhibited in FLwt cells (Fig.
2A,
) even at 12 h. This effect appears to be
modulated by cytochrome c, because addition of exogenous
cytochrome c after 3 h of permanence at 39 °C
increased greatly the levels of caspase-9 activation in ST14A cells
(Fig. 2B). Nevertheless, FLwt cells showed no caspase-9
activity even when exogenous cytochrome c was added (Fig.
2B).
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Fig. 2.
Inhibition of caspase-9 activity in FLwt
cells. The release of the fluorogenic afc moiety from the
caspase-9 substrate LEHD-afc is reported at various time points (0, 3, 9, and 12 h) following the temperature shift (A) and
before or after cytochrome c addition to the lysates
(B). A, time 0, basal levels of caspase-9
activity in normal growth conditions, i.e. 33 °C. After
exposure to SDM at 39 °C, parental ST14A cells ( ) showed an
increased caspase-9 activity. Higher activity was observed in Flmu
cells (
). In contrast, Flwt cells (
) showed no caspase-9
activity, even at 12 h after the temperature shift. Data are
expressed as nmols of cleaved substrate. One experiment is shown of
five performed on different clones that gave similar results.
B, at 3 h after the temperature shift, cellular
extracts were exposed to LEHD-afc in the presence of
cytochrome c. In treated lysates, an increased release of
the fluorogenic moiety was observed. In contrast, no caspase-9 activity
was detected in cytochrome c-triggered FLwt cells.
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Fig. 3.
Cytochrome c is found in the
cytoplasm of FLwt cells that survived. Western blot analysis was
performed on the mitochondrial and cytosolic fractions obtained from
cells grown in normal passaging conditions, i.e. 33 °C
(basal conditions) and after a 4-h incubation in SDM at 39 °C
(SDM). The protocol used and described under "Experimental
Procedures" was the one that gave the best and most reproducible
subfractionations of the four protocols utilized. Under basal
conditions, cytochrome c remained within the mitochondria in
parental (lane 1) and FLwt (lane 2) cells.
Instead, leakage of cytochrome c from the mitochondria of
Flmu cells was observed as revealed in the blot of the cytosolic
fraction (lane 3). At 4 h after the temperature shift
(SDM), cytochrome c was entirely localized to the
cytosol of all cell clones. Notably, this ability was also maintained
in FLwt cells. Tubulin reactivity in the same preparations is shown.
Column bars, the intensity of the immunoreactive
bands.
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Fig. 4.
Huntingtin inhibits cytochrome
c-dependent cleavage of caspase-9.
Cytoplasmic S-100 extracts of ST14A cells, N548wt, FLwt, N548mu, and
Flmu cells were prepared and incubated with increasing amounts of
cytochrome c, dATP, and in vitro
transcribed-translated [35S]procaspase-9. Processing of
procaspase-9 in parental ST14A cells appeared at a cytochrome
c concentration of 4 ng/µl. Importantly, in N548wt and
FLwt cells, cleavage of procaspase-9 was inhibited even at the higher
cytochrome c doses utilized. By contrast, N548mu and FLmu
cells also processed procaspase-9 at the lowest cytochrome c
concentration employed. Exogenous N548wt or N548mu proteins are present
at similar levels in the S-100 fraction.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. T. Greenamyre and Dr. A Panov for sharing their unpublished protocol on subcellular fractionation.
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FOOTNOTES |
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* This work was supported by the Huntington's Disease Society of America (H. D. S. A.), by the Hereditary Disease Foundation (H. D. F.), Telethon (E840), and Ministero dell'Universita' e della Ricerca Scientifica (MURST MM06278849-005) (to E. C.), and Universita' di Milano-Progetto Giovani (to D. R.).
These authors contributed equally to this work.
§ A member of the Coalition for the Cure (H. D. S. A.) and of the Cure Initiative (H. D. F.).
¶ To whom correspondence should be addressed. Tel.: 39-02-20488349; Fax: 39-02-29404961; E-mail: elena.cattaneo@unimi.it.
Published, JBC Papers in Press, March 5, 2001, DOI 10.1074/jbc.C100044200
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ABBREVIATIONS |
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The abbreviations used are: HD, Huntington's disease; Htt, Huntingtin; mu, mutated; wt, wild type; FL, full-length; Z-LEHD-FMK, benzyloxycarbonyl-LEHD-fluoromethylketone; mAb, monoclonal antibody; SDM, serum-depleted medium; afc, LEHD-7-amino-4-trifluoromethyl coumarin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CNS, central nervous system.
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