Expression of polyglutamine-expanded huntingtin induces tyrosine phosphorylation of N-methyl-D-aspartate receptors

In our previous studies, we found that expression of polyglutamine-expanded huntingtin in HN33 cells induced sensitization of N-methyl-D-aspartate (NMDA) receptors (Sun, Y., Savinainen, A., and Liu, Y. F. (2001) J. Biol. Chem. 276, 24713-24718). Following this study, we investigated whether tyrosine phosphorylation of NMDA receptors might contribute to the altered property of the receptors. Expression of polyglutamine-expanded huntingtin induced elevation of phosphorylated or activated Src and increased targeting of PSD-95 (post-synaptic density 95) and activated Src to cell surface membrane. Expression of the mutated huntingtin also induced tyrosine phosphorylation of NR2B (NMDA receptor 2B) subunits, and co-expression of PSD-95 enhanced the phosphorylation. Treatment of SU6656 (a specific Src inhibitor) or co-expression of a mutated NR2B subunit with mutations of all three major tyrosine phosphorylation sites significantly attenuated neuronal toxicity induced by the mutated huntingtin. Addition of AP-5 did not further inhibit the neuronal toxicity. Taken together, our studies show that polyglutamine-expanded huntingtin increases tyrosine phosphorylation of NMDA receptors via PSD-95 and Src, and increased tyrosine phosphorylation may contribute to the sensitization of the receptors mediated by polyglutamine-expanded huntingtin.     

Activation of MLK2-mediated signaling cascades by polyglutamine-expanded huntingtin

We previously reported that expression of polyglutamine-expanded huntingtin induces apoptosis via c-Jun amino-terminal kinase (JNK) activation in HN33 cells (Liu, Y. F. (1998) J. Biol. Chem. 273, 28873-28822). Extending this study, we now demonstrate a role of mixed-lineage kinase 2 (MLK2), a JNK activator, in polyglutamine-expanded huntingtin-mediated neuronal toxicity. We find that normal huntingtin interacts with MLK2, whereas the polyglutamine expansion interferes with this interaction. Similar to the expression of polyglutamine-expanded huntingtin, expression of MLK2 also induces JNK activation and apoptosis in HN33 cells. Co-expression of dominant negative MLK2 significantly attenuates neuronal apoptosis induced by the mutated huntingtin. Furthermore, over-expression of the N terminus of normal huntingtin partially rescues the neuronal toxicity induced by MLK2. Our results suggest that activation of MLK2-mediated signaling cascades may be partially involved in neuronal death induced by polyglutamine-expanded huntingtin.     

Modulation of the channel activity of the epsilon2/zeta1-subtype N-methyl D-aspartate receptor by PSD-95

A channel-associated protein PSD-95 has been shown to induce clustering of N-methyl D-aspartate (NMDA) receptors, interacting with the COOH terminus of the epsilon subunit of the receptors. The effects of PSD-95 on the channel activity of the epsilon2/zeta1 heteromeric NMDA receptor were examined by injection of PSD-95 cRNA into Xenopus oocytes expressing the NMDA receptors. Expression of PSD-95 decreased the sensitivity of the NMDA receptor channels to L-glutamate. Mutational studies showed that the interaction between the COOH terminus of the epsilon2 subunit of the NMDA receptor and the second PSD-95/Dlg/Z0-1 domain of PSD-95 is critical for the decrease in glutamate sensitivity. It is known that protein kinase C markedly potentiates the channel activity of the NMDA receptor expressed in oocytes. PSD-95 inhibited the protein kinase C-mediated potentiation of the channels. Thus, we demonstrated that PSD-95 functionally modulates the channel activity of the epsilon2/zeta1 NMDA receptor. PSD-95 makes signal transmission more efficient by clustering the channels at postsynaptic sites. In addition to this, our results suggest that PSD-95 plays a protective role against neuronal excitotoxicity by decreasing the glutamate sensitivity of the channels and by inhibiting the protein kinase C-mediated potentiation of the channels.     

The chaperonin TRiC controls polyglutamine aggregation and toxicity through subunit-specific interactions

Misfolding and aggregation of proteins containing expanded polyglutamine repeats underlie Huntington's disease and other neurodegenerative disorders. Here, we show that the hetero-oligomeric chaperonin TRiC (also known as CCT) physically interacts with polyglutamine-expanded variants of huntingtin (Htt) and effectively inhibits their aggregation. Depletion of TRiC enhances polyglutamine aggregation in yeast and mammalian cells. Conversely, overexpression of a single TRiC subunit, CCT1, is sufficient to remodel Htt-aggregate morphology in vivo and in vitro, and reduces Htt-induced toxicity in neuronal cells. Because TRiC acts during de novo protein biogenesis, this chaperonin may have an early role preventing Htt access to pathogenic conformations. Based on the specificity of the Htt-CCT1 interaction, the CCT1 substrate-binding domain may provide a versatile scaffold for therapeutic inhibitors of neurodegenerative disease.     

Kainate receptor activation induces mixed lineage kinase-mediated cellular signaling cascades via post-synaptic density protein 95

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.     

Characterization of glutamate toxicity in cultured rat cerebellar granule neurons at reduced temperature

We have defined conditions whereby glutamate becomes toxic to isolated cerebellar granule neurons in a physiologic salt solution (pH 7.4). In the presence of a physiologic Mg⁺⁺ concentration, acute glutamate excitotoxicity manifests only when the temperature was reduced from 37°C to 22°C. In contrast to glutamate, N-methyl-D-aspartate (NMDA) was non-toxic at either temperature at concentrations as high as 1 mM. Glycine strongly potentiated both the potency and efficacy of glutamate but revealed only a modest NMDA response. The non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxalinedione (CNQX), potently protected against glutamate challenge, although the contribution of antagonism at strychnine-insensitive glycine sites could not be excluded. To further characterize the non-NMDA receptor contribution to the excitotoxic response, the promiscuity of glutamate interaction with ionotropic receptors was simulated by exposing neurons to NMDA in the presence of non-NMDA receptor agonists. NMDA toxicity was potentiated four- to sevenfold when non-NMDA receptors were coactivated by a subtoxic concentration of AMPA, kainate, or domoate. These results suggest that non-NMDA receptor activation participates in the mechanism of acute glutamate toxicity by producing neuronal depolarization (via sodium influx), which in turn promotes the release of the voltage-dependent magnesium blockade of NMDA receptor ion channels. © 1997 John Wiley & Sons, Inc.     

Oligodendrocyte NMDA receptors: a novel therapeutic target

Excessive glutamate signaling can lead to excitotoxicity, a phenomenon whereby over-activation of glutamate receptors initiates neuronal death. In recent years, it has been shown that glutamate can be toxic to white-matter oligodendrocytes. Up to recently, the prevailing view was that oligodendrocyte excitotoxicity is mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate glutamate receptor types. Three recent studies have shown that oligodendrocytes also express N-methyl-D-aspartate (NMDA) receptors, which are activated under pathological conditions. Thus, NMDA receptors seem to be a promising target for the development of new drugs to treat white-matter damage in acute and chronic diseases.     

Molecular mechanisms of glutamate receptor-mediated excitotoxic neuronal cell death

Excitotoxicity is one of the most extensively studied processes of neuronal cell death, and plays an important role in many central nervous system (CNS) diseases, including CNS ischemia, trauma, and neurodegenerative disorders. First described by Olney, excitotoxicity was later characterized as an excessive synaptic release of glutamate, which in turn activates postsynaptic glutamate receptors. While almost every glutamate receptor subtype has been implicated in mediating excitotoxic cell death, it is generally accepted that the N-methyl-D-aspartate (NMDA) subtypes play a major role, mainly owing to their high calcium (Ca²⁺) permeability. However, other glutamate receptor subtypes such as 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionate (AMPA) or kainate receptors have also been attributed a critical role in mediating excitotoxic neuronal cell death. Although the molecular basis of glutamate toxicity is uncertain, there is general agreement that it is in large part Ca²⁺-dependent. The present review is aimed at summarizing the molecular mechanisms of NMDA receptor and AMPA/kainate receptor-mediated excitotoxic neuronal cell death.     

Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and nonneuronal cells

Huntington's disease is a neurodegenerative disorder caused by CAG expansion that results in expansion of a polyglutamine tract at the extreme N terminus of huntingtin (htt). htt with polyglutamine expansion is proapoptotic in different cell types. Here, we show that caspase inhibitors diminish the toxicity of htt. Additionally, we define htt itself as an important caspase substrate by generating a site-directed htt mutant that is resistant to caspase-3 cleavage at positions 513 and 530 and to caspase-6 cleavage at position 586. In contrast to cleavable htt, caspase-resistant htt with an expanded polyglutamine tract has reduced toxicity in apoptotically stressed neuronal and nonneuronal cells and forms aggregates at a much reduced frequency. These results suggest that inhibiting caspase cleavage of htt may therefore be of potential therapeutic benefit in Huntington's disease.     

Protection by cholesterol-extracting cyclodextrins: a role for N-methyl-D-aspartate receptor redistribution

Cyclodextrins (CDs) are cyclic oligosaccharides composed of a lipophilic central cavity and a hydrophilic outer surface. Some CDs are capable of extracting cholesterol from cell membranes and can affect function of receptors and proteins localized in cholesterol-rich membrane domains. In this report, we demonstrate the neuroprotective activity of some CD derivatives against oxygen-glucose deprivation (OGD), N-methyl-D-aspartic acid (NMDA) and glutamate in cortical neuronal cultures. Although all CDs complexed with NMDA or glutamate, only beta-, methylated beta- and sulfated beta-CDs displayed neuroprotective activity and lowered cellular cholesterol. Only CDs that lowered cholesterol levels redistributed the NMDA receptor NR2B subunit, PSD-95 (postsynaptic density protein 95 kDa) and neuronal nitric oxide synthase (nNOS) from Triton X-100 insoluble membrane domains to soluble fractions. Cholesterol repletion counteracted the ability of methylated beta-CD to protect against NMDA toxicity, and reversed NR2B, PSD-95 and nNOS localization to Triton X-100 insoluble membrane fraction. Surprisingly, neuroprotective CDs had minimal effect on NMDA receptor-mediated increases in intracellular Ca(2+) concentration ([Ca(2+)](i)), but did suppress OGD-induced increases in [Ca(2+)](i). beta-CD, but not Mbeta-CD, also caused a slight block of NMDA-induced currents, suggesting a minor contribution to neuroprotection by direct action on NMDA receptors. Taken together, data suggest that cholesterol extraction from detergent-resistant microdomains affects NMDA receptor subunit distribution and signal propagation, resulting in neuroprotection of cortical neuronal cultures against ischemic and excitotoxic insults. Since cholesterol-rich membrane domains exist in neuronal postsynaptic densities, these results imply that synaptic NMDA receptor subpopulations underlie excitotoxicity, which can be targeted by CDs without affecting overall neuronal Ca(2+) levels.     

Neuroprotection of GluR5-containing kainate receptor activation against ischemic brain injury through decreasing tyrosine phosphorylation of N-methyl-D-aspartate receptors mediated by Src kinase

Previous studies indicate that cerebral ischemia breaks the dynamic balance between excitatory and inhibitory inputs. The neural excitotoxicity induced by ionotropic glutamate receptors gain the upper hand during ischemia-reperfusion. In this paper, we investigate whether GluR5 (glutamate receptor 5)-containing kainate receptor activation could lead to a neuroprotective effect against ischemic brain injury and the related mechanism. The results showed that (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA), a selective GluR5 agonist, could suppress Src tyrosine phosphorylation and interactions among N-methyl-D-aspartate (NMDA) receptor subunit 2A (NR2A), postsynaptic density protein 95 (PSD-95), and Src and then decrease NMDA receptor activation through attenuating tyrosine phosphorylation of NR2A and NR2B. More importantly, ATPA had a neuroprotective effect against ischemia-reperfusion-induced neuronal cell death in vivo. However, four separate drugs were found to abolish the effects of ATPA. These were selective GluR5 antagonist NS3763; GluR5 antisense oligodeoxynucleotides; CdCl(2), a broad spectrum blocker of voltage-gated calcium channels; and bicuculline, an antagonist of gamma-aminobutyric acid A (GABA(A)) receptor. GABA(A) receptor agonist muscimol could attenuate Src activation and interactions among NR2A, PSD-95 and Src, resulting the suppression of NMDA receptor tyrosine phosphorylation. Moreover, patch clamp recording proved that the activated GABA(A) receptor could inhibit NMDA receptor-mediated whole-cell currents. Taken together, the results suggest that during ischemia-reperfusion, activated GluR5 may facilitate Ca(2+)-dependent GABA release from interneurons. The released GABA can activate postsynaptic GABA(A) receptors, which then attenuates NMDA receptor tyrosine phosphorylation through inhibiting Src activation and disassembling the signaling module NR2A-PSD-95-Src. The final result of this process is that the pyramidal neurons are rescued from hyperexcitability.     

N-Methyl-d-Aspartate Receptor Desensitisation Is Neuroprotective by Inhibiting Glutamate-Induced Apoptotic-Like Death

Abstract: Glutamate excitotoxicity is implicated in several neurodegenerative diseases; consequently, considerable effort has been made to elucidate neuroprotective mechanisms against such toxicity. N -Methyl- d -aspartate (NMDA) receptor desensitisation is one potential mechanism for controlling glutamate-mediated neuronal cell death. Pretreatment of rat cerebellar granule cells with subtoxic concentrations of NMDA caused a marked reduction in the calcium signals generated by subsequent glutamate stimulation, and, furthermore, this receptor desensitisation was coupled to a reduction in glutamate-induced apoptotic-like death. These effects were reduced by either d -2-amino-5-phosphonopentanoic acid, an NMDA receptor antagonist, or cyclosporin A, an inhibitor of calcineurin. Thus, the results support a role for receptor desensitisation in protection from glutamate-mediated apoptotic-like neuronal cell death.     

Overactivation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-D-aspartate but not kainate receptors inhibits phosphatidylcholine synthesis before excitotoxic neuronal death

Glutamate receptor overactivation induces excitotoxic neuronal death, but the contribution of glutamate receptor subtypes to this excitotoxicity is unclear. We have previously shown that excitotoxicity by NMDA receptor overactivation is associated with choline release and inhibition of phosphatidylcholine synthesis. We have now investigated whether the ability of non-NMDA ionotropic glutamate receptor subtypes to induce excitotoxicity is related to the ability to inhibit phosphatidylcholine synthesis. alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-induced a concentration-dependent increase in extracellular choline and inhibited phosphatidylcholine synthesis when receptor desensitization was prevented. Kainate released choline and inhibited phosphatidylcholine synthesis by an action at AMPA receptors, because these effects of kainate were blocked by the AMPA receptor antagonist LY300164. Selective activation of kainate receptors failed to release choline, even when kainate receptor desensitization was prevented. The inhibition of phosphatidylcholine synthesis evoked by activation of non-desensitizing AMPA receptors was followed by neuronal death. In contrast, specific kainate receptor activation, which did not inhibit phosphatidylcholine synthesis, did not produce neuronal death. Choline release and inhibition of phosphatidylcholine synthesis were induced by AMPA at non-desensitizing AMPA receptors well before excitotoxicity. Furthermore, choline release by AMPA required the entry of Ca(2+) through the receptor channel. Our results show that AMPA, but not kainate, receptor overactivation induces excitotoxic cell death, and that this effect is directly related to the ability to inhibit phosphatidylcholine synthesis. Moreover, these results indicate that inhibition of phosphatidylcholine synthesis is an early event of the excitotoxic process, downstream of glutamate receptor-mediated Ca(2+) overload.     

Protective up-regulation of CK2 by mutant huntingtin in cells co-expressing NMDA receptors

Huntington's disease is caused by a polyglutamine expansion in the huntingtin (htt) protein, and previous data indicate that over-activation of NMDA receptors (NMDARs) may be involved in the selective degeneration of cells expressing NR1/NR2B NMDARs. We used Kinetworks™ multi-immunoblotting screens to examine expression of 76 protein kinases, 18 protein phosphatases, 25 heat shock/stress proteins, and 27 apoptosis proteins in human embryonic kidney 293 cells transfected with NR1/NR2B and htt containing 15 (htt-15Q; wild-type) or 138 (htt-138Q; mutant) glutamine repeats. Follow-up experiments revealed several proteins involved in the heat-shock response pathway to be up-regulated in the soluble fraction from cells expressing htt-138Q, including protein phosphatase 5 and cyclin-dependent kinase 5. Increased expression in the soluble fraction of htt-138Q-expressing cells was also noted for the stress- and calcium-activated protein-serine/threonine kinase casein kinase 2, a change which was confirmed in striatal tissue of yeast artificial chromosome transgenic mice expressing full-length mutant htt. Inhibition of casein kinase 2 activity in cultured striatal neurons from these mice significantly exacerbated NMDAR-mediated toxicity, as assessed by labeling of apoptotic nuclei. Our findings are consistent with up-regulation of components of the stress response pathway in the presence of polyglutamine-expanded htt and NR1/NR2B which may reflect an attempt at the cellular level to ameliorate the detrimental effects of mutant htt expression.     

Effects of Mutant Huntingtin on mGluR5-Mediated Dual Signaling Pathways: Implications for Therapeutic Interventions

Glutamate excitotoxicity is thought to play an important role in Huntington’s disease (HD), which is caused by a polyglutamine expansion in the HD protein huntingtin (htt). Overactivation of group I metabotropic glutamate receptors (mGluRs), which include mGluR1 as well as mGluR5 and are coupled via phospholipase C to the inositol phosphate pathway, is found to be involved in mutant htt-mediated neurotoxicity. However, activation of mGluR5 also leads to neuronal protection. Here, we report that mutant htt can activate both mGluR5-mediated ERK and JNK signaling pathways. While increased JNK signaling causes cell death, activation of ERK signaling pathway is protective against cell death. Expression of mutant htt in cultured cells causes greater activation of JNK than ERK. These findings suggest that selective inhibition of the JNK signaling pathway may offer an effective therapeutic approach for reducing htt-mediated excitotoxicity.     

Mutant huntingtin binds the mitochondrial fission GTPase dynamin-related protein-1 and increases its enzymatic activity

Huntington's disease is an inherited and incurable neurodegenerative disorder caused by an abnormal polyglutamine (polyQ) expansion in huntingtin (encoded by HTT). PolyQ length determines disease onset and severity, with a longer expansion causing earlier onset. The mechanisms of mutant huntingtin-mediated neurotoxicity remain unclear; however, mitochondrial dysfunction is a key event in Huntington's disease pathogenesis. Here we tested whether mutant huntingtin impairs the mitochondrial fission-fusion balance and thereby causes neuronal injury. We show that mutant huntingtin triggers mitochondrial fragmentation in rat neurons and fibroblasts of individuals with Huntington's disease in vitro and in a mouse model of Huntington's disease in vivo before the presence of neurological deficits and huntingtin aggregates. Mutant huntingtin abnormally interacts with the mitochondrial fission GTPase dynamin-related protein-1 (DRP1) in mice and humans with Huntington's disease, which, in turn, stimulates its enzymatic activity. Mutant huntingtin-mediated mitochondrial fragmentation, defects in anterograde and retrograde mitochondrial transport and neuronal cell death are all rescued by reducing DRP1 GTPase activity with the dominant-negative DRP1 K38A mutant. Thus, DRP1 might represent a new therapeutic target to combat neurodegeneration in Huntington's disease.     

The role of Bax in glutamate-induced nerve cell death

The role of the Bax gene product was examined in three forms of cortical nerve cell death in primary cultures. These include spontaneous cell death, oxidative glutamate toxicity, in which exogenous glutamate inhibits cystine uptake resulting in toxic oxidative stress, and ionotropic glutamate receptor-mediated excitotoxicity following a brief exposure to 10 microM glutamate. Primary cortical and hippocampal neuron cultures were established from embryos of Bax -/+ x Bax -/+ matings and the embryos genotyped and assayed for cell death in the three experimental paradigms. Cell death induced by oxidative glutamate toxicity and glutamate-mediated excitotoxicity was not altered in the Bax -/- homozygous knockout animals. In contrast, there was an approximately 50% inhibition of spontaneous cell death. These results suggest that a classical Bax-dependent apoptotic pathway contributes to the spontaneous cell death that takes place when nerve cells are initially exposed to cell culture conditions. A Bax-dependent programmed cell death pathway is not, however, utilized in oxidative glutamate toxicity and NMDA receptor-mediated excitotoxicity following a brief exposure to low concentrations of glutamate.     

Co-chaperone CHIP associates with expanded polyglutamine protein and promotes their degradation by proteasomes

A major hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions of the disease proteins that are ubiquitinated and often associated with various chaperones and proteasome components. But, how the polyglutamine proteins are ubiquitinated and degraded by the proteasomes are not known. Here, we demonstrate that CHIP (C terminus of Hsp70-interacting protein) co-immunoprecipitates with the polyglutamine-expanded huntingtin or ataxin-3 and associates with their aggregates. Transient overexpression of CHIP increases the ubiquitination and the rate of degradation of polyglutamine-expanded huntingtin or ataxin-3. Finally, we show that overexpression of CHIP suppresses the aggregation and cell death mediated by expanded polyglutamine proteins and the suppressive effect is more prominent when CHIP is overexpressed along with Hsc70.     

Oxidative stress promotes mutant huntingtin aggregation and mutant huntingtin-dependent cell death by mimicking proteasomal malfunction

Huntington's disease (HD) is a familial neurodegenerative disorder caused by an abnormal expansion of CAG repeats in the coding region of huntingtin gene. A major hallmark of HD is the proteolytic production of N-terminal fragments of huntingtin containing polyglutamine repeats that form ubiquitinated aggregates in the nucleus and cytoplasm of the affected neurons. However, the mechanism by which the mutant huntingtin causes neurodegeneration is not well understood. Here, we found that oxidative stimuli enhance the polyglutamine-expanded truncated N-terminal huntingtin (mutant huntingtin) aggregation and mutant huntingtin-induced cell death. Oxidative stimuli also lead to rapid proteasomal dysfunction in the mutant huntingtin expressing cells as compared to normal glutamine repeat expressing cells. Overexpression of Cu/Zn superoxide dismutase (SOD1), Hsp40 or Hsp70 reverses the oxidative stress-induced proteasomal malfunction, mutant huntingtin aggregation, and death of the mutant huntingtin expressing cells. Finally, we show the higher levels of expression of SOD1 and DJ-1 in the mutant huntingtin expressing cells. Our result suggests that oxidative stress-induced proteasomal malfunction might be linked with mutant huntingtin-induced cell death.     

PSD-95 assembles a ternary complex with the N-methyl-D-aspartic acid receptor and a bivalent neuronal NO synthase PDZ domain.

Nitric oxide (NO) biosynthesis in cerebellum is preferentially activated by calcium influx through N-methyl-D-aspartate (NMDA)-type glutamate receptors, suggesting that there is a specific link between these receptors and neuronal NO synthase (nNOS). Here, we find that PSD-95 assembles a postsynaptic protein complex containing nNOS and NMDA receptors. Formation of this complex is mediated by the PDZ domains of PSD-95, which bind to the COOH termini of specific NMDA receptor subunits. In contrast, nNOS is recruited to this complex by a novel PDZ-PDZ interaction in which PSD-95 recognizes an internal motif adjacent to the consensus nNOS PDZ domain. This internal motif is a structured "pseudo-peptide" extension of the nNOS PDZ that interacts with the peptide-binding pocket of PSD-95 PDZ2. This asymmetric interaction leaves the peptide-binding pocket of the nNOS PDZ domain available to interact with additional COOH-terminal PDZ ligands. Accordingly, we find that the nNOS PDZ domain can bind PSD-95 PDZ2 and a COOH-terminal peptide simultaneously. This bivalent nature of the nNOS PDZ domain further expands the scope for assembly of protein networks by PDZ domains.