From the Department of Psychiatry and Behavioral Neurobiology,
University of Alabama at Birmingham, Birmingham,
Alabama 35294-0017
Received for publication, June 12, 2002, and in revised form, November 3, 2002
Huntington's disease (HD) is an autosomal
dominant neurodegenerative disorder caused by an abnormally expended
polyglutamine domain. There is no effective treatment for HD; however,
inhibition of caspase activity or prevention of mitochondria
dysfunction delays disease progression in HD mouse models. Similarly
administration of cystamine, which can inhibit transglutaminase,
prolonged survival of HD mice, suggesting that inhibition of
transglutaminase might provide a new treatment strategy. However, it
has been suggested that cystamine may inhibit other
thiol-dependent enzymes in addition to transglutaminase. In
this study we show that cystamine inhibits recombinant active caspase-3
in a concentration-dependent manner. At low
concentrations cystamine is an uncompetitive inhibitor of caspase-3
activity, becoming a non-competitive inhibitor at higher
concentrations. The IC50 for cystamine-mediated
inhibition of caspase-3 activity in vitro was 23.6 µM. In situ cystamine inhibited in a
concentration-dependent manner the activation of caspase-3
by different pro-apoptotic agents. Additionally, cystamine inhibited
caspase-3 activity to the same extent in cell lines stably
overexpressing wild type tissue transglutaminase (tTG), a mutant
inactive tTG, or an antisense for tTG, demonstrating that cystamine
inhibits caspase activity independently of any effects it may have on
the transamidating activity of tTG. Finally, treatment with cystamine
resulted in a robust increase in the levels of glutathione. These
findings demonstrate that cystamine may prolong neuronal survival and
delay the onset of HD by inhibiting caspases and increasing the level
of antioxidants such as glutathione.
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INTRODUCTION |
Huntington's disease
(HD)1 is an autosomal
dominant neurodegenerative disorder caused by an abnormal expansion of
a CAG repeat in the gene encoding for huntingtin, a protein of unknown
function (1). Although, the precise mechanisms responsible for the
pathogenesis of HD remain to be elucidated, there is evidence to
suggest that the genetic defect in huntingtin is associated with
mitochondrial dysfunction leading to an increase in oxidative stress
conditions (2). Further, recent studies support a role for caspases in the progression of HD (3, 4), and aberrant protein-protein interactions
related to the abnormally expanded polyglutamine stretches likely play
a critical role in the etiology of HD (5, 6). Indeed, the presence of
neuronal intranuclear and cytoplasmic inclusions composed of mutant
huntingtin in HD brain constitutes a striking neuropathological
hallmark of the disease (7, 8). However, the role of the aggregates in
HD pathogenesis remains to be elucidated. Another feature of HD brain
is a significant increase in tissue transglutaminase (tTG) (9, 10), and
it has been hypothesized that tTG may contribute to the etiology of
several neurodegenerative disorders such as HD (9).
Tissue TG (also called type 2 TG) belongs to a family of
thiol-dependent transamidating enzymes that catalyze a
calcium-dependent acyl transfer reaction between the
-carboxamide group of a polypeptide-bound glutamine and the
-amino group of a polypeptide-bound lysine residue to form an
-(
-glutamyl) lysine isopeptide bond (for review see Ref. 9).
Transglutaminases can also catalyze the incorporation of a polyamine
leading to the formation of a (
-glutamyl)lysine isopeptide bond
(11). The recent resolution of the x-ray crystallographic structure of
human tTG has defined its structural and functional domains (12).
Tissue TG is organized into four domains: a
-sandwich domain,
followed by a transamidation catalytic core domain, and two
carboxyl-terminal
-barrel domains (12, 13). The catalytic core of
tTG comprises a catalytic triad constituted of the residues cysteine,
histidine, and aspartic acid (Cys-277, His-335, and Asp-358 in human
tTG) (14). This catalytic triad is conserved in all members of the TG
family and presents remarkable similarity with the papain-like
catalytic center (13, 15), suggesting a common evolutionary lineage
(13), and providing the structural features suggesting that the
mechanism of the TGs is similar to the reverse mechanism of the
cysteine proteinases (15). Given the fact that a polypeptide-bound
glutamine is the primary determining factor for a tTG-catalyzed
reaction it has been suggested that tTG may contribute to the formation
of huntingtin aggregates in HD (16). Indeed, tTG levels and TG activity
are significantly elevated in brain area affected in HD (10, 17). These
findings have raised the hypothesis that inhibition of TG may be an
effective therapeutic strategy for HD. However, in a recent study we
demonstrated that tTG is not necessary to the formation of mutant
huntingtin aggregates (18). Further, treatment of HD mouse with the TG inhibitor cystamine did not affect the appearance or the frequency of
neuronal inclusions (19). Nonetheless, cystamine treatment has
beneficial effects in an HD mouse model (19). Because cystamine inhibits tTG, likely by forming a mixed disulfide, it has been suggested that, in addition to inhibiting TG, cystamine might interfere
with and inhibit other thiol-dependent enzymes such as the
caspases (20). This hypothesis is particularly relevant in the context
of HD as inhibition of caspases delays the disease progression in HD
mouse models (3, 4). Therefore, the purpose of this study was to
further analyze the properties of cystamine and particularly to
determine whether cystamine inhibits caspase activity and thereby
contributes to the protective effects that have been described in
cellular (21) and mouse models of polyglutamine disorders (19).
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EXPERIMENTAL PROCEDURES |
Materials--
Cystamine was obtained from Sigma, the
glutathione reductase (lot B43049) and MG132 were purchased from
Calbiochem, and the caspase substrate AC-DEVD-AMC was obtained from
Alexis. The purified recombinant active human caspase-3 (CPP32) (lot
MO62912) and the monoclonal antibody for poly(ADP-ribose) (PARP) were
obtained from BD Pharmingen/Transduction Laboratories. The monoclonal
tTG antibody TG100 was from Neomarkers.
Cell Culture--
Human neuroblastoma SH-SY5Y cells were
maintained in RPMI 1640 media supplemented with 20 mM
glutamine, 10 units/ml penicillin, 100 µg/ml streptomycin, 5% fetal
clone II serum (HyClone), and 10% horse serum. Cells were maintained
in a humidified 37 °C incubator with 5% CO2. SH-SY5Y
cells that stably overexpress tTG, mutated inactive tTG (C277S), or
antisense tTG were described previously (22, 23). Stably transfected
cells were maintained in the same media containing 100 µg/ml G418
(Geneticin). SH-SY5Y cells were plated at a density of
~105 cells/60-mm dish 48 h before
apoptosis-inducing treatments. To determine the effects of cystamine on
caspase-3 activity, cells were placed in serum-free media and incubated
in the absence of cystamine or in the presence of various
concentrations of cystamine for 10 h prior to treatment with
apoptotic stressors. Cells were subsequently treated with the indicated
concentrations of the proteosome inhibitor MG132 for 16 h, which
previously has been shown to induce apoptosis in various cell types
(24, 25), or with 100 µM hydrogen peroxide for the time
indicated prior to measurement of caspase-3 activation or PARP cleavage.
Cell Viability--
The release of the intracellular enzyme
lactate dehydrogenase (LDH) into the medium was used as a quantitative
measurement of cell viability. The measurement of LDH was carried out
as described previously (26). The percentage of LDH released was
defined by LDH activity in the medium divided by total LDH activity.
Collection of Cell Lysates--
Cells were collected in the
media and spun at 2000 × g at 4 °C for 10 min. Cell
pellets were washed twice in ice-cold phosphate-buffered saline prior
to being resuspended in lysis buffer containing 50 mM
Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA,
0.5% Nonidet P-40, 0.1 mM phenylmethylsulfonyl fluoride,
and a 10 µg/ml concentration of each of aprotinin, leupeptin, and
pepstatin, sonicated on ice and spun at 2000 × g at
4 °C for 10 min. Protein concentration of the supernatant was
determined using the bicinchoninic acid assay (BCA) method (Pierce).
Immunoblotting--
Cell lysates were diluted to a final
concentration of 1 mg/ml in 2× reducing stop buffer (0.25 M Tris-HCl (pH 7.5), 2% SDS, 25 mM
dithiothreitol, 5 mM EGTA, 5 mM EDTA, 10%
glycerol, and 0.01% bromphenol blue as tracking dye) and incubated in
a boiling water bath for 5 min. Proteins were separated on 7.5 and 10%
for immunoblotting PARP and tTG, respectively. The proteins were then transferred to nitrocellulose. Blots were probed with monoclonal antibodies to PARP and tTG, followed by incubation with horseradish peroxidase-conjugated secondary antibody. Blots were developed using
peroxidase substrate chemiluminescence (Amersham Biosciences).
In Situ Caspase Activity--
In situ caspase-3
activity was measured using a previously described protocol (27). In
brief, 200 µl of assay buffer (20 mM Hepes, pH 7.5, 10%
glycerol, and 2 mM dithiothreitol) containing the peptide
substrate for caspase-3 (AC-DEVD-AMC) was added to each well (final
concentration of 25 ng/µl) of a 96-well clear bottom plate (Corning).
Cell lysate (20 µg of protein) was added to start the reaction.
Triplicate measurements were done for each sample. Background
fluorescence was measured in wells containing assay buffer, substrate,
and lysis buffer without the cell lysate. Assay plates were incubated
at 37 °C for 1 h, and fluorescence was measured on a
fluorescence plate reader (Bio-Tek, Winooski, VT) set at 360 nm
excitation and 460 nm emission.
In Vitro Caspase Activity--
Purified recombinant active human
caspase-3 was incubated in 20 mM PIPES (pH 7.2), 100 mM NaCl, 1 mM EDTA, 0.1% CHAPS, and 10%
sucrose (total volume 200 µl). The reaction was started by adding
to each well the peptide substrate for caspase-3 (AC-DEVD-AMC). When
the effects of cystamine on caspase-3 activity were examined, cystamine
was added at the concentrations indicated immediately before addition
of the caspase-3 substrate.
Measurement of Glutathione Levels--
Cells (60-mm plate,
~0.2-0.4 mg of protein) were washed twice with ice-cold phosphate
buffered saline and deproteinized with 200 µl of ice-cold 5% (w/v)
5-sulfosalicylic acid (SSA). Forty microliters of a 1 M
HEPES/1 M KOH solution was added to protein-free SSA, and
the pH was adjusted to 7.0 with 1 M KOH. Total glutathione (GSH + GSSG) was measured by an enzymatic recycling procedure in which
it was sequentially oxidized by 5,5'-dithiobis-(2-nitrobenzoic acid)
and reduced by NADPH in the presence of glutathione reductase (28). The
rate of 2-nitro-5-thiobenzoic formation was monitored at 412 nm, and
the concentration of glutathione determined by comparison of that
result with a standard curve generated with known amounts of GSH.
Statistic Analysis--
Data were analyzed using Student's
t test. Values were considered significantly different when
the two-tailed p value was <0.05. Results are expressed as
means ± S.E.
 |
RESULTS |
Cystamine Inhibits Caspase-3 Activity in Vitro--
Cystamine is
known to inhibit TG activity (29), probably by forming a mixed
disulfide, raising the possibility that cystamine may react in a
similar manner with other thiol-dependent enzymes such as
the caspases (20). To test this hypothesis, the activity of human
recombinant caspase-3 (5-20 ng) was measured in the absence or
presence of various concentrations of cystamine (0-500
µM) (Fig. 1, A
and B). Under these experimental conditions, recombinant caspase-3 in the absence of cystamine exhibited a
concentration-dependent increase in activity (Fig.
1A) and the caspase-3-mediated cleavage of AC-DEVD-AMC curve
was within a linear range (r = 0.97) over a time course
of at least 60 min (Fig. 1B). To examine the effects of
cystamine the activity was measured using different amounts of
recombinant active caspase-3 (5-20 ng) incubated in the presence of
increasing concentrations of cystamine (31.2-500 µM).
The results from a typical experiment are shown in Fig. 1 and revealed
that cystamine inhibited caspase-3 activity in a
concentration-dependent manner (Fig. 1, A and
B). Caspase-3 activity was significantly decreased (~60%)
in the presence of 31.2 µM cystamine and almost completely inhibited in the presence of 500 µM cystamine
(Fig. 1, A and B). The IC50 for
cystamine-mediated inhibition of caspase-3 activity was 23.6 µM. A Lineweaver-Burk analysis (Fig.
2) demonstrated that cystamine is an
uncompetitive inhibitor of caspase-3 activity, with a
Ki of 38.5 µM; however, at the highest
concentration (500 µM) cystamine became a non-competitive
inhibitor of caspase-3 activity. Thus, in vitro cystamine is
an inhibitor of caspase-3 activity.

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Fig. 1.
Cystamine inhibits caspase-3 activity
in vitro. The activity of recombinant human
caspase-3 incubated in the absence or presence of various
concentrations of cystamine was measured using the caspase-3 substrate
DEVD-AMC. A, recombinant active caspase-3 (5-20 ng) was
incubated for 1 h at 37 °C in the absence or presence of
cystamine (0-500 µM) prior to measuring caspase-3
activity. Incubation in the presence of cystamine resulted in a
concentration-dependent inhibition of caspase-3 activity;
the IC50 for cystamine inhibition of caspase-3 activity was
23.6 µM. Results are expressed as arbitrary units of
fluorescence (AUF), and representative data from a typical
experiment are shown (n = 4 independent experiments and
each measurement was done in triplicate). B, recombinant
caspase-3 (20 ng) was incubated in the absence or presence of various
concentrations of cystamine (0-500 µM) for 0-60 min. In
the absence of cystamine, caspase-3 activity was within the linear
range (r = 0.97) over a time course of at least 60 min.
Representative data from a typical experiment are presented
(n = four independent experiments, and each measurement
was done in triplicate). The concentration of cystamine is indicated at
the right of each line.
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Fig. 2.
Lineweaver-Burk plot of data from the
cystamine-mediated inhibition of recombinant caspase-3 activity.
Cystamine acts as an uncompetitive inhibitor of caspase-3 activity,
becoming a noncompetitive inhibitor at higher concentrations
(n = 4 independent experiments; representative data of
a typical experiment are presented). The concentration of cystamine is
indicated for each line.
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|
Cystamine Does Not Alter Cell Viability--
The effects of
cystamine on caspase-3 activity were examined next in situ.
Prior to measuring the effects of cystamine on caspase-3 activity
in situ, the effects of cystamine on cell viability were
analyzed by measuring LDH release. Incubation of SH-SY5Y cells with
cystamine (31.2-500 µM) for 24 h did not result in a significant increase in LDH release compared with cells exposed to
the vehicle alone, revealing that cystamine did not induce a
significant loss in cell viability at these concentrations (data not shown).
Effect of Cystamine on MG132-induced Caspase Activation--
To
examine the effects of cystamine on caspase-3 activity in
situ, SH-SY5Y cells were pre-incubated with or without cystamine (31.2-500 µM) for 10 h prior to incubation in the
absence or presence of the proteasome inhibitor MG132 (200 nM) for 16 h. Treatment of SH-SY5Y cells with MG132
alone resulted in a significant increase in caspase-3 activity compared
with cells treated with vehicle alone (Fig.
3A). The MG132-mediated
increase in caspase activity was further confirmed by the
caspase-dependent proteolysis of PARP (Fig. 3B).
Incubation of SH-SY5Y cells with cystamine (31.2-500 µM)
prior treatment with MG132 prevented in a dose-dependent
manner the activation of caspase-3 (Fig. 3A). The
MG132-mediated increase in caspase-3 activity was significantly
attenuated by 125 µM cystamine and completely inhibited
by 500 µM cystamine (Fig. 3A). The finding that cystamine inhibited caspase-3 activity induced by MG132 was further supported by a reduced degree of PARP proteolysis in cells incubated with cystamine prior to treatment with MG132 (Fig.
3B).

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Fig. 3.
Cystamine inhibits the MG132-mediated
activation of caspase-3. A, cells were incubated in the
absence or presence of the indicated concentrations of cystamine for
10 h before treatment with MG132 (200 µM) for
16 h; cells were then collected, and caspase-3 activity measured.
Treatment with MG132 resulted in a significant increase in caspase-3
activity as compared with cells exposed to the vehicle alone.
Pretreatment of the cells with cystamine resulted in a
concentration-dependent inhibition of the MG132-mediated
increase in caspase-3 activity. Results are expressed as a percent of
caspase-3 activity compared with cells exposed to the vehicle alone;
means ± S.E. for four independent experiments; *,
p < 0.05 compared with cells treated with MG132 alone.
B, the caspase-dependent proteolytic cleavage of
PARP was measured by immunoblotting lysates from cells that have been
incubated in the absence or presence of cystamine (500 µM) prior to treatment with the indicated concentrations
of MG132 (0-500 µM) for 16 h. Pretreatment of
SH-SY5Y cells with cystamine potently prevented the MG132-mediated
caspase cleavage of PARP to the 85-kDa fragment. Representative
immunoblot from a typical experiment are shown (n = four independent experiments).
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Effect of Cystamine on H2O2-induced Caspase
Activation--
To further examine the effect of cystamine on
caspase-3 activity another apoptotic paradigm was used:
H2O2-induced increases in caspase-3 activity.
In the absence of cystamine, 100 µM
H2O2 treatment resulted in an increase in
caspase-3 activity starting at 2 h after
H2O2 treatment and reaching a maximum after
6 h (Fig. 4A). The
time-dependent proteolytic cleavage of PARP to the 85-kDa fragment after treatment with 100 µM
H2O2 corresponded to the time-dependent increase in caspase-3 activity (Fig.
4B). Pretreatment of SH-SY5Y cells with cystamine (250 µM) significantly attenuated the increase in caspase-3
activity mediated by treatment with H2O2 (Fig.
4A). The effect of cystamine on inhibiting the
H2O2-mediated increase in caspase-3 activity
were further confirmed by the reduced degree of caspase-mediated PARP
proteolysis in cells that had been pretreated with cystamine prior to
H2O2 treatment (Fig. 4B). Thus,
in situ cystamine significantly prevents the activation caspase-3 mediated by two different apoptotic stressors. Further, treatment of SH-SY5Y cells with cystamine completely prevented the
increase in caspase-3 activity resulting from the withdrawal of trophic
factors from the media (data not shown), revealing that the effect of
cystamine in preventing the increase in caspase-3 activity did not
result from a direct effect of cystamine on the proapoptotic agents
used to treat the cells but rather from an effect in the cell,
presumably by acting on caspase-3.

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Fig. 4.
Cystamine inhibits the
H2O2-mediated activation of caspase-3.
SH-SY5Y cells were incubated in the absence or presence of cystamine
(250 µM) for 10 h before treatment with
H2O2 (100 µM) for 0-8 h.
A, cells were then collected, and caspase-3 activity
measured. Results are expressed as a percent of caspase-3 activity in
cells exposed to the vehicle alone; means ± S.E. for three to
five independent experiments; *, p < 0.05 compared
with cells exposed to H2O2 alone at each time
point. B, PARP proteolysis was examined in cell lysates by
immunoblotting. The H2O2-mediated proteolytic
cleavage of PARP was greatly decreased in cells incubated with
cystamine prior to exposure to H2O2.
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Cystamine Inhibits Caspase Activity Independently of
tTG--
Cystamine is known to inhibit tTG activity (29); therefore we
next examined the potential contribution of the cystamine-induced inhibition of tTG on the cystamine-prevented activation of caspase-3. For this, the effects of cystamine on caspase-3 activity were examined
in cell lines stably overexpressing the wild type tTG, mutated inactive
C277S tTG, an antisense for tTG, and as a control, the empty vector.
These different cell lines have been extensively characterized in
previous studies (22, 23). The levels of tTG expression in the
different cell lines used in this experiment are shown in Fig.
5A. The level of tTG
expression was low in cells stably transfected with the empty vector
(pcDNA) and not detected in cells stably transfected with the
antisense tTG (Fig. 5A). However, in cells stably expressing
wild type tTG (tTG cells) or mutant inactive tTG (C277S cells), tTG
expression was significantly higher, and similar expression levels were
detected in both cell lines (Fig. 5A). In the four different
cell lines treatment with MG132 (200 nM for 16 h)
resulted in a significant increase in caspase-3 activity compared with
cells treated with the vehicle alone (data not show). Pretreatment of
the different cell lines with cystamine (250 µM) prior
exposure to MG132 almost completely prevented the MG132-mediated
activation of caspase-3 activity (Fig. 5B), and cystamine
inhibited caspase-3 activity to the same extent for all the cell lines
demonstrating that cystamine inhibits the activation of caspase-3
independently of any effects it may have on the transamidating activity
of tTG.

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Fig. 5.
Cystamine inhibits caspase-3 activity in a
tTG-independent manner. A, representative immunoblot of
the levels of tTG in the different cell lines. In SH-SY5Y cells stably
transfected with the empty vector (Vect) or with an
antisense for tTG (Anti) the level of tTG protein was low or
undetectable, respectively. However in cell stably transfected with the
wild type human tTG (tTG) or with the mutant inactive tTG (C277S) a
significant and equivalent increase in tTG expression was observed.
B, to determine the contribution of tTG to the
cystamine-mediated inhibition of caspase, caspase-3 activity was
measured in the different cell lines that were incubated in the absence
or presence of cystamine (250 µM) prior to incubation in
the absence or presence of MG132 (200 nM, 16 h). In
the four cell lines pretreatment with cystamine inhibited the
MG132-mediated increase in caspase-3 activity to the same extent
revealing that cystamine inhibits caspase-3 activity independently of
tTG. Data presented are means ± S.E. (n = five to
six independent experiments, each in triplicate; *, p < 0.05).
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Treatment with Cystamine Results in an Increase in Glutathione
Level--
Because it has been previously suggested that cystamine
treatment may affect the state of the glutathione system (30, 31), the
effects of cystamine on the level of glutathione in the cell were
examined. SH-SY5Y cells were incubated in the absence or presence of
250 µM cystamine for 8 h prior to collection and
measurement of the levels of glutathione (GSH + GSSG) (Fig.
6). In the absence of cystamine the level
of glutathione in the cells was 12.7 ± 4.2 nmol/mg of protein.
Treatment with cystamine significantly increased the levels of
glutathione to 40.2 ± 11.04 nmol/mg of protein (Fig. 6).

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Fig. 6.
Treatment with cystamine results in an
increase in glutathione levels. SH-SY5Y cells were incubated in
the presence (+) or absence ( ) of cystamine (250 µM)
for 10 h, and the level of glutathione (GSH + GSSG) was measured.
Cystamine treatment resulted in a significant increase in glutathione
levels as compared with cells treated with the vehicle alone. Data are
presented as means ± S.E. (n = five independent
experiments; *, p < 0.05).
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 |
DISCUSSION |
Tissue TG is a multifunctional enzyme that has been implicated in
several physiological processes and pathological conditions (for review
see Ref. 9). Indeed, recent evidence suggests that a dysregulation of
tTG may contribute to the pathology of polyglutamine disorders such as
HD (9, 16, 32). The potential role of tTG in the etiology of HD and
other polyglutamine diseases is particularly intriguing as it has been
suggested that tTG may contribute to the formation of insoluble nuclear
and cytoplasmic aggregates that constitute a striking neuropathological
hallmark of many of these diseases including HD (7, 8). However, in a
recent study we demonstrated that modulating the level and activity of
tTG did not affect the frequency or localization of huntingtin
aggregates, revealing that tTG was not necessary for their formation
(18). In support of these findings, ablation of tTG in an HD mouse
model (R6/1 mice) resulted in a significant increase in the frequency
of huntingtin aggregates (33). Further, administration of cystamine,
initially characterized as a TG inhibitor, to 7-week-old transgenic
mice expressing exon 1 of huntingtin containing an expanded
polyglutamine repeat (R6/2 mice) did not influence the appearance or
frequency of neuronal nuclear inclusions (19). These results strongly
suggest that tTG is not necessary for aggregate formation in HD;
however, in the R6/2 mice cystamine treatment was beneficial as it
prolonged survival of the mice and decreased the abnormal movements and
associated tremor (19). Further in a cell culture system, cystamine
decreased the apoptotic cell death induced by the expression of a
truncated dentatotubral-pallidoluysian atrophy protein containing an
expanded polyglutamine repeat domain (21). Based on the effects of
cystamine, it has been suggested that inhibition of tTG may provide a
new treatment strategy for HD (19). However, it has been hypothesized
that cystamine inhibits tTG by forming a mixed disulfide in the active
site, raising the possibility that a similar reaction may also occur
with other thiol-dependent enzymes such as caspases (20).
Indeed, the results of this study unequivocally demonstrate that
in vitro and in situ cystamine inhibits caspase-3
activity in a tTG independent manner. However, at low concentrations
cystamine acts as an uncompetitive inhibitor of caspase-3, revealing
that cystamine preferentially inhibits caspase-3 after formation of the
complex enzyme-substrate, rather than preventing interaction of the
substrate with caspase-3. This suggests that the interaction of
caspase-3 with a substrate causes a conformational change in caspase-3
allowing cystamine to interact with the target thiol group. Cystamine
by inhibiting caspase-3 may contribute to the observed neuroprotective
effects of cystamine in the HD mouse model. Several studies have
demonstrated that caspases may play a critical role in the etiology of
HD. For example, lymphoblasts derived from HD patients showed increased stress-induced apoptotic cell death associated with caspase-3 activation (34). Similarly treatment of R6/2 mice with the
tetracycline-derivative drug minocycline inhibits caspase-1 and
caspase-3 expression and delays disease progression (3). Further, the
combined inhibition of both caspase-1 and caspase-3 is required for
effective pharmacotherapy in the R6/2 mouse model of HD (3). Finally,
crossbreeding the R6/2 mice with transgenic mice expressing a
dominant-negative mutant of caspase-1 in the brain extended the
survival and delayed neurotransmitter receptor alterations and onset of
the symptoms (4). These studies strongly suggest that in the R6/2 mice
caspases may play an important role in the etiology of the disease (4). Considering the potential implication of caspases in HD etiology, it is
likely that the cystamine-mediated inhibition of caspase-3, and
potentially other caspases, could contribute to the prolonged survival
observed in HD mouse models.
Our results show that cystamine treatment of SH-SY5Y cells induces a
robust increase in the intracellular level of glutathione, which plays
a key role in protecting cells against oxidative damage by reacting
with hydrogen peroxide and organic peroxides. There is compelling
evidence to demonstrate that the HD gene defect is associated with
impaired oxidative phosphorylation, resulting in an increase in
oxidative damage (35-37). Indeed, biochemical analysis of postmortem
brain tissues provided evidence of impaired mitochondrial enzyme
activity in brain regions affected in HD (37). Further, increased
oxidative damage is also well documented in HD brains (38); these
include increased evidence of DNA strand break and increased levels of
oxidative damage products such as 8-hydroxydeoxyguanosine,
3-nitrotyrosine, and malondialdehyde in brain regions affected in HD
(38). These findings suggest that a neuroprotective strategy for HD
might include the prevention of mitochondria dysfunction and the
resulting oxidative damage. Indeed, dietary creatine supplementation
significantly improved survival and slowed the formation of
huntingtin-positives aggregates in HD mouse models (39, 40).
Considering these and others findings, it can be hypothesized that by
increasing the level of antioxidants such as glutathione and inhibiting
caspases, cystamine may prolong neuronal survival and contribute to the
delay in the onset of the disease.
We thank Dr. P. J. A. Davies (University of
Texas Medical School) for providing the cDNA coding for the human
tTG and Dr. R. S. Jope (University of Alabama at Birmingham) and
members of his laboratory for helpful advice with the experiments using
H2O2.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M205812200
The abbreviations used are:
HD, Huntington's
disease;
TG, transglutaminase;
tTG, tissue transglutaminase;
PARP, poly(ADP-ribose);
LDH, lactate dehydrogenase;
PIPES, 1,4-piperazinediethanesulfonic acid;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
SSA, 5-sulfosalicylic acid.
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