Kainate Receptor Activation Induces Mixed Lineage Kinase-mediated Cellular Signaling Cascades via Post-synaptic Density Protein 95*

Anneli SavinainenDagger , Elizabeth P. Garcia§, Donna Dorow, John Marshall§, and Ya Fang LiuDagger ||

From the Dagger  Department of Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115, the § Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, Rhode Island 02912, and the  Trecowthick Research Center, Peter MacCallum Cancer Institute, Melbourne, Victoria 3000, Australia

Received for publication, January 9, 2001



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Kainate receptor glutamate receptor 6 (GluR6) subunit-deficient and c-Jun N-terminal kinase 3 (JNK3)-null mice share similar phenotypes including resistance to kainite-induced epileptic seizures and neuronal toxicity (Yang, D. D., Kuan, C-Y., Whitmarsh, A. J., Rincon, M., Zheng, T. S., Davis, R. J., Rakis, P., and Flavell, R. (1997) Nature 389, 865-869; Mulle, C., Seiler, A., Perez-Otano, I., Dickinson-Anson, H., Castillo, P. E., Bureau, I., Maron, C., Gage, F. H., Mann, J. R., Bettler, B., and Heinemmann, S. F. (1998) Nature 392, 601-605). This suggests that JNK activation may be involved in GluR6-mediated excitotoxicity. We provide evidence that post-synaptic density protein (PSD-95) links GluR6 to JNK activation by anchoring mixed lineage kinase (MLK) 2 or MLK3, upstream activators of JNKs, to the receptor complex. Association of MLK2 and MLK3 with PSD-95 in HN33 cells and rat brain preparations is dependent upon the SH3 domain of PSD-95, and expression of GluR6 in HN33 cells activated JNKs and induced neuronal apoptosis. Deletion of the PSD-95-binding site of GluR6 reduced both JNK activation and neuronal toxicity. Co-expression of dominant negative MLK2, MLK3, or mitogen-activated kinase kinase (MKK) 4 and MKK7 also significantly attenuated JNK activation and neuronal toxicity mediated by GluR6, and co-expression of PSD-95 with a deficient Src homology 3 domain also inhibited GluR6-induced JNK activation and neuronal toxicity. Our results suggest that PSD-95 plays a critical role in GluR6-mediated JNK activation and excitotoxicity by anchoring MLK to the receptor complex.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Glutamate, the major excitatory neurotransmitter in the central nervous system, gates three types of ionotropic receptors: NMDA,1 AMPA, and kainate (3). Five kainate receptor subunits in two homology groups have been identified: KA1, KA2, and GluR5, GluR6, and GluR7 (4, 5). Expression of individual GluR5-7 subunits in heterologous systems results in homomeric receptors that respond to glutamate or kainic acid with a rapidly desensitizing current (5, 6). KA1 and KA2, on the other hand, are functional only when coexpressed with GluR5, -6, or -7 (5, 6).

PSD-95, also known as synapse-associated protein 90, is a scaffold protein that contains three PDZ domains, a SH3 domain, and a guanylate kinase domain (7). The PDZ domains have been shown to bind to the C terminus of NMDA receptor NR2 and kainate receptor GluR6 subunits, and these interactions are important for the clustering of NMDA or kainate receptors in the postsynaptic membrane (7-9). In addition, PSD-95 also binds to cytoskeletal linker proteins and cytoplasmic signaling proteins such as neuronal nitric oxide synthase and the Src family protein tyrosine kinase Fyn (10, 11). PSD-95 appears to link NMDA or kainate receptors to a variety of cellular signaling cascades. In transgenic mice lacking PSD-95, although the localization of NMDA receptors at post-synaptic density remains unaltered, the frequency dependence of NMDA-dependent long-term potentiation and long-term depression is shifted, and spatial learning is severely impaired (12). Suppression of PSD-95 expression inhibits NMDA receptor-mediated activation of nitric-oxide synthase and neuronal excitotoxicity (13), which suggests that PSD-95 is critical in coupling glutamate receptors to cellular signaling networks and plays an important role in their biological function within the central nervous system.

JNK is a major stress-activated kinase in mammalian systems that is implicated in mediating neuronal death induced by various detrimental stimuli and by glutamate-mediated excitotoxicity (14-16). In the absence of JNK3, a neuronal form of JNK, kainic acid-induced seizure activity and neuronal degeneration are significantly attenuated (1). This phenotype is strikingly similar to that observed in GluR6-deficient mice (2), which suggests that JNK activation may be involved in GluR6-mediated excitotoxicity.

Both MLK2 and MLK3 are members of the mixed lineage kinase family typified by a N-terminal SH3 domain, a middle kinase domain, and a C-terminal proline-rich region that may bind to SH3 domain-containing proteins. MLK2/3 can directly bind and activate MKK4 and MKK7, which in turn phosphorylate and activate JNKs (17-18). Studies from our group and others show that expression of MLK2 induces JNK activation and apoptotic cell death (18-19).

The current study was undertaken to investigate the molecular mechanism of GluR6-mediated excitotoxicity. We hypothesized that the SH3 domain of PSD-95 binds to the proline-rich region of MLK2 or MLK3 and recruits these kinases to the proximity of GluR6, leading to JNK activation and neuronal apoptosis.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Cell culture conditions for HN33 cells, an immortalized rat hippocampal neuronal cell line, and 293T cells have been described previously (20). c-Myc-tagged full-length MLK2 was a generous gift of Dr. A. Hall (University College of London, United Kingdom) and Flag-tagged full-length MLK3 was a gift from Dr. A. Rana (Massachusetts General Hospital, Boston, MA). HN33 or 293T cells at 50 to 60% confluence were washed once with serum-free medium prior to transfection. Transfection was performed using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's instructions. 10-20 µg of plasmids with 10 µl of LipofectAMINE/10-cm plate was used in transfection experiments.

Co-immunoprecipitation and in Vitro Binding Assays-- Antibodies for PSD-95 and GluR6 were purchased from Upstate Biotechnology Inc., and M2, 9E10, and MLK3 antibody were purchased from Santa Cruz. A peptide selected from the MLK2 sequence (amino acids 248-265 DFGLAREWHKTTKMSAAG) was conjugated to KLH for immunization of rabbits, and the resulting polyclonal antibody was purified. The antibody recognizes a 105-110-kDa protein band as expected for MLK2 in the rat brain lysates and the 9E10 immunoprecipitates of 293T cells transfected with c-Myc-tagged MLK2, but is not found in wild-type 293T cell lysates, which lack MLK2. This protein band is no longer detectable when the antibody was pre-absorbed with the peptide antigen (data not shown). Preparation of cell or whole brain lysates and co-immunoprecipitation were conducted as described previously (9, 21). Precipitated proteins were analyzed by Western blotting using an antibody as indicated in the figure legends. A Kodak 440 Image Station was used to analyze and quantify blots. Construction and purification of different PSD-95 GST fusion proteins have been described previously (9). 1-2 µg of GST fusion protein was used for in vitro binding studies.

JNK and TUNEL Assays-- 16 h after transfection, HN33 cells were lysed with 1% Triton X-100 lysis buffer (20). JNK was assayed as described previously (19, 20). 24 to 48 h following transfection, cells were fixed and TUNEL staining was performed as previously described (19, 20). Most apoptotic HN33 cells were detached from the slides and TUNEL staining was performed on the remaining attached cells. TUNEL negative cells (i.e. living cells) were counted in the ×20 power field at four different locations, and 600-800 cells were counted in the control.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To test our hypothesis, we first examined whether MLK2 or MLK3 was associated with PSD-95 in 293T cells coexpressing c-Myc-tagged MLK2 or Flag-tagged MLK3. Wild-type 293T cells which lack PSD-95, served as a negative control. 9E10 or M2 antibodies, which detect the c-Myc or Flag epitopes, respectively, were incubated with 293T cell lysates and immunoprecipitated proteins were analyzed by an anti-PSD-95 monoclonal antibody. As shown in Fig. 1A, PSD-95 was detected in 9E10 or M2 immunoprecipitates only from 293T cell lysates co-transfected with MLK2/3 and PSD-95 but was not detected from wild-type cell lysates. Next, we examined whether MLK2 or MLK3 is associated with PSD-95 in HN33 cells or in the rat brain. PSD-95 was detected in MLK2/3 immunoprecipitates from HN33 cell lysates or whole rat brain lysates but not in precipitates obtained with preimmune serum or peptide-antigen preabsorbed MLK2 antibody (Fig. 1B). Conversely, immunoprecipitates of PSD-95 also contained MLK2 (Fig. 1C) or MLK3 (Fig. 1D).



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Fig. 1.   Association of PSD-95 with MLK2 or MLK3. All data presented represent a typical experiment that has been repeated 2-3 times with similar results. A, full-length PSD-95 and c-Myc-tagged MLK2 or Flag-tagged MLK3 constructs were transiently expressed in 293T cells. Cell lysates were immunoprecipitated with 9E10 or M2 and the blot was analyzed by an anti-PSD-95 antibody. 9E10W or M2W, immunoprecipitated with wild-type 293T cell lysates; 9E10T or N2T, immunoprecipitated with transfected 293T cell lysates. B, detection of PSD-95 in MLK2 or MLK3 immunoprecipitates: MLK2-pre, brain lysates immunoprecipitated with MLK2 preimmune serum; MLK2-ab, brain lysates immunoprecipitated with peptide-antigen preabsorbed MLK2 antibody; BrainMLK3, HN33MLK2, or BrainMLK2, brain or HN33 cell lysates immunoprecipitated with an anti-MLK3 or anti-MLK2 antibody. C, detection of MLK2 in PSD-95 immunoprecipitates: as indicated in the figure, HN33 cell or brain lysates were immunoprecipitated with an anti-PSD-95 antibody and the blot was analyzed by an anti-MLK2 antibody. D, detection of MLK3 in PSD-95 immunoprecipitates using HN33 cell or brain lysates. E, detection of GluR6 in MLK2 or MLK3 immunoprecipitates but not in the preimmune serum of the MLK2 antibody.

These data indicate that PSD-95 is associated with MLK2/3 in neuronal cells and rat brain tissues. PSD-95 is known to bind to GluR6 (9). Because MLK2/3 is associated with PSD-95, they may be indirectly associated with GluR6 in the brain. As shown in Fig. 1E, GluR6 was detected in both MLK2 and MLK3 immunoprecipitates but not precipitates obtained with preimmune serum.

We next explored the molecular basis for the association of PSD-95 with MLK2/3. Both MLK2 and MLK3 contain a proline-rich region that may bind to the SH3 domain of PSD-95. PSD-95 GST fusion proteins containing PDZ1 (amino acids 2-151), PDZ2 (amino acids 156-266), PDZ3 (amino acids 302-401), SH3 (amino acids 402-500), GK (amino acids 512-724), or SH3-GK (SGS, amino acids 402-724) domain were constructed and subsequently purified (9). These GST fusion proteins were incubated with c-Myc-tagged MLK2 or Flag-tagged MLK3 expressed in 293T cells. We found that MLK2 only bound to PSD-95 SH3 or SH3-GK domain (SGS) GST fusion proteins but not to any of PSD-95 PDZ GST fusion proteins or to GST alone (Fig. 2). We also observed that MLK3 bound only to the SH3 domain but not to PDZ or GK domains of PSD-95 (data not shown).



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Fig. 2.   The SH3 domain of PSD-95 mediates its binding to MLK2. c-Myc-tagged MLK2 or Flag-tagged MLK3 expressed in 293T cells were incubated with ~2 µg of GST alone or different PSD-95 GST fusion proteins and the blot was analyzed by an anti-PSD-95 antibody. 95SH3, 95SGS, 95GK, 95PDZ1, 95PDZ2, or 95PDZ3 indicates the SH3, SH3-GK, GK, PDZ1, -2, or -3 domain GST fusion proteins. The data represent a typical experiment that has been repeated three times with similar results.

The above data suggest that GluR6, PSD-95, and MLK2/3 may exist in a complex. Because MLK2/3 is a strong activator of JNKs and both JNK and GluR6-deficient mice are resistant to kainic acid-induced seizures and neuronal degeneration (1, 2), we reasoned that GluR6-mediated excitotoxicity might be mediated by the MLK-JNK pathway. Thus, we investigated whether expression of GluR6 induces JNK activation in HN33 cells. Twenty-four hours following transfection, cells were treated with 100 µM kainic acid for 5 min and JNK activity was analyzed by an anti-phospho-JNK antibody. Expression of vector alone (negative control) did not induce JNK activation, whereas expression of GluR6 caused an elevated JNK activity, which was further increased upon stimulation with 100 µM kainic acid (Fig. 3A). To eliminate the possibility that JNK activation was due to activation of other ionotropic glutamate receptors, we employed a series of receptor antagonists. Addition of 10 µM CNQX, a kainate and AMPA receptor antagonist, to the transfection medium significantly attenuated JNK activation while addition of 10 µM GYKI 52466, a selective AMPA receptor antagonist, had no effect. Addition of (-)-AP-5, a selective NMDA receptor antagonist caused a weaker inhibition of GluR6-mediated JNK activation. Coaddition of 10 µM (-)-AP-5 and CNQX to the medium nearly completely blocked JNK activation (Fig. 3A). These data suggest that activation of GluR6 largely accounts for elevated JNK activity in HN33 cells.



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Fig. 3.   Activation of JNK by GluR6. 20 h following transfection, wild-type or GluR6 transfected HN33 cell lysates were analyzed for JNK activity using an anti-phospho-JNK antibody. The data is from a typical experiment that has been repeated at least three times. A, activation of GluR6 accounts for JNK activation in HN33 cells. Different receptor antagonists as indicated in the figure were added to the transfection medium and 20 h post-transfection, cells were stimulated with 100 µM kainic acid for 5 min followed by analysis of JNK activity. GluR6Delta , GluR6 with deleted C-terminal four amino acids. B, dominant negative MLK2/3 or MKK4 and MKK7 or SH3 domain-deficient PSD-95 significantly inhibits JNK activation induced by GluR6. MLK2K/E, MLK3K/E, or mMKK4/7, dominant negative form of kinases; mPSD-95, PSD-95 (W470A).

Because MLK2/3 is a strong activator of JNK (17, 18), we next examined whether either of these two kinases are involved in GluR6-mediated JNK activation. As shown in Fig. 3B, coexpression of dominant negative MLK2 or MLK3 or MKK4 and MKK7 significantly inhibited GluR6-induced JNK activation in HN33 cells. Dominant negative MLK3 appeared to be less effective. This may be due to its lower abundance in HN33 cells. These data suggest that GluR6 may activate MLK2/3-mediated cellular signaling cascades in HN33 cells. We believe that GluR6 anchors and activates MLK2/3 via a complex involving PSD-95. If this hypothesis is correct, then either deletion of the PSD-95-binding site of GluR6 or mutation of the SH3 domain of PSD-95 should disrupt assembly of the GluR6·PSD-95·MLK2/3 complex and block JNK activation mediated by the receptor. As shown in Fig. 3A, deletion of the C-terminal four amino acids of GluR6 (GluR6Delta ) nearly completely blocked JNK activation mediated by the receptor. Co-expression of PSD-95 with a mutated SH3 domain (W470A) also significantly inhibited GluR6-mediated JNK activation (Fig. 3B).

Next, we investigated whether expression of GluR6 induces apoptotic cell death in HN33 cells. Expression of GluR6 induced rapid apoptosis, and TUNEL-positive cells were detectable 24 h following transfection. At 48 h post-transfection, about 30-35% of cells were apoptotic (Fig. 4A). Addition of a nonspecific caspase inhibitor, zDEVD-frm, to the transfection medium completely blocked neuronal death (Fig. 4A), supporting that cell death is apoptotic. PSD-95 has been implicated in activation of nNOS by NMDA receptors (13). To test the role of nNOS in GluR6-mediated neuronal toxicity in HN33 cells, Nomega -propylarginine, a selective nNOS inhibitor, was added to the transfection medium. As shown in Fig. 4A, the nNOS inhibitor had almost no effect on cell death, suggesting that nNOS does not contribute to GluR6-mediated neuronal toxicity in HN33 cells. Addition of 500 µM kainic acid to the medium 24 h following transfection significantly enhanced neuronal toxicity: the number of apoptotic cells was increased to 60% at 48 h post-transfection, compared with 40% without kainic acid (Fig. 4B). Addition of 10 µM CNQX significantly attenuated neuronal toxicity: over 80% of cells survived (Fig. 4B). Consistent with the data from JNK activation, GYKI 52466 had no effect while coaddition of 10 µM (-)-AP5 with CNQX nearly completely blocked neuronal toxicity (Fig. 4B). These data indicate that GluR6 plays a major role in the induction of neuronal toxicity in HN33 cells.



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Fig. 4.   GluR6 mediates apoptotic cell death in HN33 cells. HN33 cells were transfected with vector alone (mock) or different plasmids as indicated in the figures. 48 h post-transfection, cells were fixed and TUNEL staining was carried out and TUNEL-negative cells were counted. The data are the average of three independent experiments. Con, control. A, GluR6-mediated apoptosis was blocked by a nonspecific caspase inhibitor zDEVD-frm but not by a selective nNOS inhibitor Nϖ-propyl-L-arginine. B, activation of GluR6 mostly accounts for apoptosis. Individual receptor antagonist was added into the transfection medium and 500 µM kainic acid was added into the medium 24 h post-transfection. C, HN33 cells were co-transfected with dominant negative form of MLK2 (MLK2K/E) or MKK4 and -7 (mMKK4/7) or PSD-95W470A (mPSD-95) as indicated in the figure followed by TUNEL assay. D, deletion of the C-terminal four amino acids of GluR6 (GluR6Delta ) inhibits its ability to induce apoptosis in HN33 cells.

Next, we investigated whether activation of MLK2/3-mediated signaling cascades is involved in GluR6-mediated neuronal toxicity in HN33 cells. As shown in Fig. 4C, expression of a mutated GluR6 that does not bind to PSD-95 did not cause significant cell death, and addition of kainic acid did not enhance the toxicity. Co-expression of dominant negative MLK2 or MKK4 and MKK7 significantly inhibited apoptosis induced by GluR6 (Fig. 4D), suggesting that MLK2-mediated signaling cascades do play a significant role in the induction of neuronal toxicity. Co-expression of dominant negative MLK3 had a smaller inhibitory effect (data not shown) while co-expression of PSD-95 with a deficient SH3 domain inhibited GluR6-mediated apoptotic cell death, although the extent of the inhibition was much weaker than that mediated by dominant negative MLK2 or MKK4 and MKK7 (Fig. 4D). These data indicate that GluR6 activates MLK2/3-mediated cellular signaling cascades via PSD-95 to mediate apoptosis in HN33 cells.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GluR6 activation induces neuronal degeneration in hippocampus (2, 22), and GluR6-deficient mice exhibit a resistance to neuronal degeneration and seizure induced by kainic acid (2). A similar phenotype was also observed in JNK3 knockout mice (1), suggesting that activation of JNKs may be involved in excitotoxicity mediated by GluR6. Although some reports have shown that stimulation of kainate receptors mediates JNK activation (23), it is unclear how GluR6 mediates JNK activation and whether inhibition of JNK activation blocks GluR6-mediated neuronal toxicity. In this study, we provide evidence that GluR6 activates JNKs via the PSD-95-MLK2/3-signaling pathway. Expression and activation of GluR6 causes JNK activation and apoptosis in HN33 cells. Co-expression of dominant negative MLK2 or MLK3 significantly inhibits both JNK activation and neuronal toxicity induced by the receptor. Additionally, co-expression of dominant negative MKK4 and MKK7, which are immediate upstream activators of JNKs, also significantly inhibited GluR6-mediated JNK activation and apoptosis. MLK2 or MLK3 is associated with PSD-95 and GluR6 in intact neuronal cells and in rat brain tissues, suggesting that MLK2 or MLK3 may assemble into a signaling complex with PSD-95 and GluR6. Deletion of the PSD-95-binding site of GluR6 significantly abolished the ability of the receptor to induce JNK activation and apoptosis in HN33 cells, suggesting that binding of PSD-95 to the receptor is necessary for neuronal toxicity to occur. The SH3 domain of PSD-95 mediates its binding to MLK2/3; and mutation of the SH3 domain of PSD-95 significantly attenuates both JNK activation and apoptosis mediated by GluR6, suggesting that binding of MLK2/3 to the SH3 domain of PSD-95 is critical for induction of neuronal toxicity. MLK2/3 is a constitutively active kinase (17, 18). Our explanation is that assembly of the GluR6·PSD-95·MLK2/3 complex occurs upon activation of the receptor and promotes exposure of the kinase domain to ATP and activation of the downstream signaling pathway.

PSD-95 has been implicated in excitotoxicity and the induction of activation of nNOS by NMDA receptors (13). In HN33 cells, nNOS activation does not contribute to GluR6-induced neuronal toxicity because the nNOS inhibitor has no effect on apoptosis induced by the receptor. Some reports suggest that p38 MAPK is involved in the neuronal toxicity mediated by NMDA receptors (24). We found that addition of SB 203580, a selective p38 MAPK inhibitor, to the transfection medium had no any effect on GluR6-induced neuronal death. In addition, GluR6 failed to activate MAPK in HN33 cells. These data are consistent with the fact that, at moderate expression levels, MLK2/3 selectively activates the MKK4/7-JNK pathway (17, 18).

Kainate receptor-mediated excitotoxicity may play an essential role in the pathogenesis of intra-striatal injection of kainic acid in rats creates HD-like pathology (25). The number of kainate receptors is significantly reduced and high affinity receptors are almost absent in the brains of HD patients and HD-transgenic mice (26, 27). Moreover, the TAA repeat polymorphism of the GluR6 gene has been linked to a younger onset of HD (28, 29). In a previous report, we showed that activation of MLK2-mediated signaling cascades may contribute to neuronal death induced by polyglutamine-expanded huntingtin. The current study suggests that GluR6 activation and mutated huntingtin may share a similar molecular mechanism to induce neuronal death. This would help to explain the potential role of the receptor in the pathogenesis of HD. In summary, our present study gives new insight into how PSD-95 links a kainate receptor to stress-activated kinase signaling cascades.


    ACKNOWLEDGEMENTS

We thank Drs. R. Deth and S. Grate for helpful suggestions and critical reading of the manuscript. We express our gratitude to Dr. A. Rana for the MLK3 vector and Dr. R. Davis for MKK7 vectors.


    FOOTNOTES

* This work is supported by United States Army Medical Research and Materiel Command under cooperative agreement 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: Northeastern University, 312 Mugar Hall, 360 Huntington Ave., Boston, MA 02115. Tel.: 617-373-3904; Fax: 617-373-8886; E-mail: yafliu@lynx.neu.edu.

Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M100190200


    ABBREVIATIONS

The abbreviations used are: NMDA, N-methyl-D-aspartate; GluR6, glutamate receptor 6; MLK, mixed lineage kinase; PSD-95, post-synaptic density protein 95 (also known as SAP-90, synapse-associated protein 90); JNK, c-Jun N-terminal kinase; SH3, Src homology 3; AMPA, alpha -amino-3-hydroxy-5-methyl-4-isoxazole proprionate; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; MKK, mitogen-activated kinase kinase; HD, Huntington's disease GST, glutathione S-transferase; TUNEL, Tdt-mediated dUTP-biotin nick end labeling; KA, kainate acid; MAPK, mitogen-activated protein kinase.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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