From the Department of Pharmaceutical Sciences,
Northeastern University, Boston, Massachusetts 02115 and the
§ Neurological Science Institute, Oregon Health Science
University, Beaverton, Oregon 97006
Received for publication, April 19, 2001
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ABSTRACT |
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Increased glutamate-mediated excitotoxicity seems
to play an important role in the pathogenesis of Huntington's
disease (Tabrizi, S. J., Cleeter, M. W., Xuereb, J., Taaman,
J. W., Cooper, J. M., and Schapira, A. H. (1999)
Ann. Neurol. 45, 25-32). However, how polyglutamine
expansion in huntingtin promotes glutamate-mediated excitotoxicity
remains a mystery. In this study we provide evidence that (i) normal
huntingtin is associated with
N-methyl-D-aspartate (NMDA) and kainate
receptors via postsynaptic density 95 (PSD-95), (ii) the SH3 domain of
PSD-95 mediates its binding to huntingtin, and (iii) polyglutamine
expansion interferes with the ability of huntingtin to interact with
PSD-95. The expression of polyglutamine-expanded huntingtin
causes sensitization of NMDA receptors and promotes neuronal apoptosis
induced by glutamate. The addition of the NMDA receptor antagonist
significantly attenuates neuronal toxicity induced by glutamate and
polyglutamine-expanded huntingtin. The overexpression of normal
huntingtin significantly inhibits neuronal toxicity mediated by NMDA or
kainate receptors. Our results demonstrate that polyglutamine expansion
impairs the ability of huntingtin to bind PSD-95 and inhibits
glutamate-mediated excitotoxicity. These changes may be essential for
the pathogenesis of Huntington's disease.
Huntington's disease
(HD)1 is a dominant inherited
neurodegenerative disorder characterized by choreiform movement,
psychiatric disturbance, and cognitive decline (2). The HD gene encodes a 350-kDa protein designated as huntingtin (3), which is richly expressed in dendrites and nerve terminals, where huntingtin is associated with synaptic vesicles and microtubule complexes (4-5). The
defect of the HD gene is the expansion of a CAG repeat encoding polyglutamine at its 5' end, and the length of the repeat is correlated with the age of onset and the severity of the disease (6).
Although the HD gene has been identified for many years, how
polyglutamine-expanded huntingtin causes neurons to die remains unclear. Increased glutamate-mediated excitotoxicity in HD has been a
very popular hypothesis for the last 25 years (1). The hypothesis is
generated from findings that the intrastriatal injection of glutamate
or kainic acid in rat causes selective loss of medium spiny neurons
that are also selectively affected in HD (7-8). This hypothesis is
supported further by the findings that NMDA receptors are hyperactive,
and excitotoxicity mediated by these receptors is enhanced
significantly in HD transgenic mice (9-10). These results suggest that
overactivation of glutamate receptors may play a significant role in
the pathogenesis of HD. However, over 95% of normal or
polyglutamine-expanded huntingtin is located in the cytoplasm (6-7),
whereas glutamate receptors are cell surface receptors. How a
cytoplasmic protein alters glutamate receptors on the cell surface
membrane is an intriguing question.
PSD-95 is a scaffold protein that contains an SH3 domain, a GK domain,
and three PDZ domains that bind to the NMDA receptor NR2 subunits and
kainate receptor GluR6 subunit (11-12). The binding of PSD-95 to NMDA
or kainate receptors causes the clustering of the receptors in the
postsynaptic membrane and regulates NMDA-dependent long
term potentiation and long term depression (12). PSD-95 also binds to
cytoplasmic signaling proteins and links the receptors to cellular
signaling cascades (13-14). In transgenic mice lacking PSD-95, the
frequency function of NMDA-dependent long term potentiation and long term depression is shifted, and spatial learning is impaired severely (15). Suppression of PSD-95 expression inhibits NMDA receptor-mediated activation of nitric-oxide synthase and
excitotoxicity (16). These studies suggest that PSD-95 regulates
glutamate receptor-mediated excitotoxicity and plays an important role
in spatial learning, which is severely impaired in HD patients and HD
transgenic mice (17-19). In previous studies, we observed that the
overexpression of polyglutamine-expanded huntingtin caused neuronal
apoptosis via activation of the mixed lineage kinase/c-Jun N-terminal
kinase signaling pathway (20-21), and mixed lineage kinase is also
involved in neuronal toxicity mediated by GluR6 receptors via
interaction with PSD-95 (22). The present study is intended to
investigate the "missing link" protein between glutamate receptors
and huntingtin and to determine its pathological significance in
HD.
Cell Culture and Transient Transfection--
HN33 (an
immortalized rat hippocampal neuronal cell line) (20) and 293T cells
(human embryonic kidney cells expressing SV40 large T antigen) were
maintained in Dulbecco's modified Eagle's medium/F-12 or Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum.
Transfection was performed in serum- and Mg2+-free medium
using Lipofectin (Life Technologies, Inc.) according to the
manufacturer's instructions. Between 10 and 50 µg of plasmids with
10-20 µl of Lipofectin/10-cm plate were used in transfection experiments.
Western Blotting and Immunoprecipitation--
48-72 h after
transfection, 293T cells were harvested and lysed in 1% Nonidet P-40
lysis buffer, and co-immunoprecipitation experiments were conducted
as described previously (23). Human cortex tissues were obtained from
Dr. J.-P. Vonsattel or the Human Brain Bank at McLean Hospital
(Belmont, MA) with institutional review board approval.
Post-mortem time was between 16 and 24 h. The diagnosis of HD was
confirmed with neuropathological and genetic phenotype analysis. Human
cortex tissues from normal subjects or HD patients were homogenized in
detergent-free lysis buffer, and SDS was added to the final
concentration of 2%. The mixture was diluted 1:5 with 1% Nonidet P-40
lysis buffer. After removal of the insoluble fractions by
centrifugation, brain lysates were co-immunoprecipitated with 437, a
rabbit polyclonal antibody against the first 17 amino acids of
huntingtin that has been previously characterized (23), or other
antibodies as indicated in the figure legends.
Purification of GST Fusion Proteins and in VitroBinding
Assay--
The construction of various PSD-95 and the N terminus GST fusion
proteins of huntingtin has been described previously (21-22). Escherichia coli (DH1 TUNEL Staining--
24 or 48 h after transfection, HN33
cells were fixed with 4% paraformaldehyde and then permeabilized with
0.1% Triton X-100 for 2 min on ice, and TUNEL staining was performed
as described previously (21). Most apoptotic HN33 cells were detached
from the slides, and TUNEL staining was performed on the remaining attached cells. TUNEL stain-negative cells (living cells) were counted
in the ×20 power field in four different locations on the slides and
~600-800 cells were counted in the control (21).
Initially, we studied the interaction of normal huntingtin with
PSD-95 in 293T cells, which are rich in huntingtin (23). Full-length
PSD-95 expressing vector was transiently transfected in 293T cells.
48 h post-transfection, cell lysates were prepared, and
co-immunoprecipitation was conducted using 437, an anti-huntingtin antibody (23), or a monoclonal antibody specific for PSD-95. The
resulting blot was then probed with the anti-PSD-95 antibody. As shown
in Fig. 1A, PSD-95 was
detected in both 437 or PSD-95 (positive control) immunoprecipitates of
293T cell lysates transfected with PSD-95 but not in 437 immunoprecipitates of wild-type or vector-transfected 293T cell
lysates. We then explored the association of huntingtin with PSD-95 in
human cortex tissues. Brain lysates were immunoprecipitated with 437 or
negative control, protein A-Sepharose beads alone, 437 pre-immune
serum, or 437 pre-absorbed with its peptide antigen. The blot was
analyzed with an anti-PSD-95 antibody. As shown in Fig. 1B,
PSD-95 was only detected in 437 immunoprecipitates but not in any of
the negative controls. Conversely, huntingtin was also present in
PSD-95 immunoprecipitates (Fig. 1C). The data suggest that
huntingtin is associated with PSD-95 in human cortex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) carrying a pGEX-PSD-95 or
pGEX-huntingtin vector were grown in LB medium, and the expression of
GST fusion proteins was induced by 1 mM
isopropyl-
-thiogalactopyranoside. GST fusion proteins were recovered
by glutathione-Sepharose beads. About 1-2 µg of these GST fusion
proteins were used for in vitro binding studies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Association of huntingtin with PSD-95.
All the data presented are from a typical experiment that has been
repeated at least twice with similar results. A, the
association of huntingtin with PSD-95 in 293T cells. Lysates from
wild-type 239T cells (W) or lysates transfected with
pcDNA1 vector (V) or full-length PSD-95 (95)
were precipitated with 437 or an anti-PSD-95 antibody (PSD)
as indicated. The blot was analyzed with the anti-PSD-95 antibody.
B, the association of huntingtin with PSD-95 in human
cortex. Lysates from human cortex tissues were incubated with 437, beads alone, 437 pre-immune serum (437.Pre), or
peptide-antigen pre-absorbed 437 (437.P.ab), respectively,
and the blot was probed with an anti-PAD-95 antibody.
C, the association of huntingtin with glutamate receptors in
human cortex tissues. Lysates from human cortex tissues were incubated
with 437 pre-immune serum (437.Pre), anti-different
receptor, or anti-PSD-95 antibody as indicated. The blot was probed
with 437.
PSD-95 is known to bind to the C terminus of the NMDA receptor NR2 subunit and the kainate receptor GluR6 subunit (11-12). If huntingtin is associated with PSD-95, it may also assemble complexes with NMDA and GluR6 receptors. Thus, we investigated whether huntingtin is associated with NMDA and kainate receptors. Human cortex lysates were incubated with anti-NR1, -NR2A, -NR2B, -GluR6, or -dopamine D4 receptor or with 437 or 437 pre-immune serum. The blot was probed with 437. As shown in Fig. 1C, huntingtin was found in the anti-NR1, -NR2A, -NR2B, -GluR6, and 437 immunoprecipitates but not in the anti-D4 receptor and 437 pre-immune serum precipitates. The data suggest that huntingtin assembles complexes with NMDA and GluR6 receptors in human cortex.
Because PSD-95 has a type II SH3 domain, we examined whether normal
huntingtin can bind to the SH3 domain of PSD-95. GST alone or various
PSD-95-GST fusion proteins were prepared; 1-2 µg of these PSD-95-GST
fusion proteins were incubated with wild-type 293T cell lysates rich in
normal huntingtin. As shown in Fig. 2,
huntingtin only binds to the SH3 or SH3-GK domain, but it does not bind
to GST alone, the GK domain, or any of the three PDZ domains of
PSD-95. The data suggest that the SH3 domain of PSD-95 mediates its
binding to huntingtin.
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We next determined whether expansion of the polyglutamine repeat in
huntingtin would alter its interaction with PSD-95. The N-terminal
proline-rich region adjacent to the polyglutamine repeat has been
reported to bind to SH3 domain-containing proteins (21, 24). To
determine whether expansion of the polyglutamine repeat in huntingtin
would alter its interaction with PSD-95, we examined the binding of
PSD-95 to the huntingtin N terminus containing either a normal or
expanded polyglutamine stretch. The huntingtin N terminus GST fusion
proteins containing 16 or 56 polyglutamine repeats were generated and
purified as described previously (21). These GST fusion proteins were
incubated with 293T cell lysates expressing full-length PSD-95. As
shown in Fig. 3A, PSD-95 binds to the huntingtin N terminus containing 16 polyglutamine repeats. Because the proline region is the only SH3 domain-binding site in this
small N-terminal segment of huntingtin, these data suggest that the
N-terminal proline region of huntingtin mediates its interaction with
PSD-95. The amount of PSD-95 bound to the huntingtin N terminus with 56 polyglutamine repeats was significantly reduced, ~70% less than that
associated with normal huntingtin (Fig. 3A). This finding
suggests that polyglutamine expansion inhibits the ability of
huntingtin to bind to the SH3 domain of PSD-95.
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Next, we explored the association of huntingtin with PSD-95 in the brains of HD patients. Lysates of human cortex tissues from two normal subjects and two HD patients with mid-age onset were prepared. These lysates were incubated with 437, and the resulting blots were analyzed with an anti-PSD-95 antibody. As shown in Fig. 3B, huntingtin was associated with PSD-95 in the cortex tissues of both normal subjects and HD patients. However, the association of huntingtin with PSD-95 in HD patients is significantly weaker than that in normal subjects. Although it might be expected that the heterozygous nature of the huntingtin in HD patients would result in a PSD-95 associated with huntingtin of 50% compared with normal subjects; the amount of PSD-95 associated with huntingtin in the HD patients is much less, ~80% less than that in normal subjects (not 50% lower as one would expect, given the heterozygous nature of these HD patients). The brain lysates were precipitated with anti-PSD-95, and the blot was probed with 437. Normal huntingtin in the brains of HD patients remained to interact with PSD-95; however, the amount of huntingtin proteins associated with PSD-95 was below 50% (Fig. 3C). We also found that polyglutamine-expanded huntingtin is not present in the anti-PSD-95 immunoprecipitates in the brains of HD patients (Fig. 3C). The data suggest that polyglutamine-expanded huntingtin fails to bind to PSD-95 and that the normal huntingtin in the brains of these patients may be re-distributed. Similar results were obtained with three other normal subjects, and HD patients with different post-mortem times obtained similar results (data not shown). We conclude that the difference in the extent of the association of huntingtin with PSD-95 between normal subjects and HD patients is not because of the difference of post-mortem time but reflects a genuine change in the ability of huntingtin to bind to PSD-95 in HD patients.
The physiological relevance of the association of huntingtin with
PSD-95 was examined because PSD-95 is known to regulate NMDA receptor-
or GluR6-operated channels by clustering these receptors at
post-synaptic membranes (11-12). Our hypothesis is that normal
huntingtin modulates these receptor-operated channels by binding and
sequestering PSD-95 and thereby regulates the clustering of these
receptors. Because the ability of huntingtin to bind to PSD-95 is
impaired severely upon its polyglutamine expansion, more PSD-95
proteins may be available to cluster NMDA receptors, leading to
overactivation or sensitization of these receptors and excitotoxicity.
If our hypothesis is correct, the expression of polyglutamine-expanded
huntingtin may enhance, and overexpression of normal huntingtin may
inhibit, neuronal toxicity mediated by NMDA or GluR6 receptors. In our
previous studies, we observed that the overexpression of
polyglutamine-expanded huntingtin in HN33 cells (which expresses both
NR1 and NR2A receptors (data not shown)) induces apoptotic cell death
(20-21). To test our hypothesis, we examined whether expression of the
mutated huntingtin may promote activation of NMDA receptors in HN33
cells. Full-length huntingtin containing 16 or 48 polyglutamine repeats
(pFL16HD or pFL48HD) was transiently expressed in HN33 cells; 1 mM glutamate, 10 µM D-AP5 (a
selective NMDA antagonist), or glutamate + D-AP5 was included in the transfection medium. 24 or 48 h post-transfection, cells were fixed, TUNEL stain-detecting was conducted on
apoptotic cells, and TUNEL-negative cells (living cells) were counted.
Consistent with our previous reports, HN33 cells transfected with
pFL48HD began to undergo apoptosis at ~24 h post-transfection (Fig.
4A). At 48 h, ~60% of
HN33 cells were apoptotic (Fig. 4A). However, the treatment
of wild-type HN33 cells or cells transfected with pFL16HD with
glutamate did not alter cell viability (Fig. 4A). Also,
treatment of HN33 cells transfected with pFL48HD with glutamate significantly promoted neuronal toxicity. At 24 h
post-transfection, ~35-40% of HN33 cells were apoptotic when
glutamate was included in the transfection medium, compared with
10-15% that were apoptotic when glutamate was not included in the
transfection medium without glutamate (Fig. 4A). At 48 h, 85% of HN33 cells were apoptotic when treated with glutamate as
compared with ~60% of apoptotic cells in the glutamate-free
transfection medium (Fig. 4A). The addition of
D-AP5 significantly attenuated neuronal toxicity mediated by both glutamate and the mutated huntingtin. At 48 h, the number of apoptotic cells induced by the mutated huntingtin alone was reduced
to 30% and in the presence of D-AP5, the number of
apoptotic cells induced by both mutated huntingtin and glutamate was
reduced to 36% (Fig. 4B). The data suggest that the
expression of polyglutamine-expanded huntingtin may sensitize and
activate NMDA receptors, leading to neuronal toxicity.
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Our hypothesis predicts that overexpression of the normal huntingtin N
terminus (which binds and sequesters PSD-95) may inhibit neuronal
toxicity induced by both mutated huntingtin and NMDA receptors. To test
this possibility, pFL48HD was co-transfected with pN16HD (truncated
huntingtin encoded by the first three exons with 16 polyglutamine
repeats) into HN33 cells, and 1 mM glutamate was added to
the transfection medium. As shown in Fig. 4C, the number of
apoptotic cells induced by glutamate and mutated huntingtin was
significantly reduced when pN16HD was co-expressed. This
neuroprotective action mediated by normal huntingtin was abolished when
full-length PSD-95 was co-introduced (Fig. 4C). The data
support the view that normal huntingtin sequesters PSD-95 and thereby
inhibits neuronal toxicity mediated by glutamate receptors.
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DISCUSSION |
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Striatal medium spiny neurons are the first population of neurons affected in HD. It seems that the activation of NMDA or kainate receptors in the striatum also selectively induces the death of the same population despite the fact that these receptors are widely distributed in other types of striatal neurons (25). Mice transgenic for the HD gene exhibit hyperactive NMDA receptors in the brain and are deficient in long term depression and spatial learning (9-10), suggesting that expression of the mutated huntingtin can cause sensitization and activation of membrane NMDA receptors. However, full-length polyglutamine-expanded huntingtin displays perinuclear localization (26-27). PSD-95 is known to bind and regulate the activity of glutamate receptors (11-12). Thus, the association of huntingtin with PSD-95 clearly provides a crucial link between glutamate receptors and the mutated huntingtin.
Our current study indicates that normal huntingtin binds to PSD-95 and sequesters the scaffold protein, resulting in the inhibition of NMDA receptor activity. Overexpression of the normal huntingtin N terminus significantly attenuates neuronal toxicity induced by both NMDA receptors and the mutated huntingtin, and co-expression of wild-type PSD-95 inhibits the neuroprotective action of normal huntingtin. This suggests that PSD-95 is a mediator of neuronal toxicity induced by NMDA receptors and mutated huntingtin. The data are consistent with our recent report (23), as well as studies from other groups (16). Gain of function has been a popular hypothesis in the HD field in the last 20 years (28). Our previous and current studies show that loss of function may play an important role in the induction of neuronal death in HD (21). Consistent with our results, other groups have also shown that the expression of normal huntingtin in HD transgenic mice attenuates neuronal toxicity induced by the mutated huntingtin (29). Thus, the competitive loss of function of normal huntingtin may initiate the pathogenesis of HD. An interesting question is why heterozygous patients exhibit a similar HD phenotype with homozygous patients. We found that the ability of normal huntingtin to bind to PSD-95 in HD patients is also impaired and that the amount of huntingtin associated with the scaffold protein is well below 50%. In other words, the mutated huntingtin not only fails to bind to PSD-95 but also inhibits the binding of normal huntingtin to the scaffold protein in the human post-mortem brains. These observations reflect the dominant nature of the huntingtin mutation and may explain the clinical similarity between homozygous and heterozygous HD patients (29).
Clinically, HD is characterized by movement disorders,
cognitive decline, and psychiatric disturbance (4, 17). If a protein were essential for the pathogenesis of HD, one would assume that disruption of the biological function of this protein might lead to
these clinical symptoms. PSD-95 fits the role of such a protein because
PSD-95 is known to be involved in excitotoxicity mediated by glutamate
receptors (16, 22). Increased glutamate-mediated excitotoxicity plays a
critical role in neuronal loss in HD (1-3, 9-10). Gene-targeted
knockout of PSD-95 severely impairs spatial learning, which is also a
feature of HD patients and transgenic mice (17-19). The alteration of
NMDA receptor-mediated function causes schizophrenic behavior
(30), and many HD patients have schizophrenia-like psychiatric
disturbances (4, 17). Thus, our present study may reflect an important
molecular mechanism underlying pathological changes in HD.
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ACKNOWLEDGEMENT |
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We thank Drs. S. Grate, I. Ebong, and G. Perides for critical reading and helpful comments on this manuscript.
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FOOTNOTES |
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* This work was supported by the United States Army Medical Research and Materiel Command under cooperative agreement No. DAMD17-00-2-0012 (to Y. F. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Pharmacology, Boston University, 715 Albany St., Boston, MA 02118-2526. Tel.: 617-638-4300; Fax: 617-638-4329; E-mail: yafliu@bu.edu.
Published, JBC Papers in Press, April 23, 2001, DOI 10.1074/jbc.M103501200
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ABBREVIATIONS |
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The abbreviations used are: HD, Huntington's disease; NMDA, N-methyl-D-aspartate; PSD-95, post-synaptic density 95; SH3, src-homology 3; GK, guanylate kinase; NR, NMDA receptor; GluR6, glutamate receptor 6; GST, glutathione S-transferase; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP biotin nick end labeling; D-AP5, 3-amino-5-phosphonovalerate.
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