Selective knockdown of gene expression of N-methyl-D-aspartate receptor one ameliorates parkinsonian motor symptom in 6-hydroxydopamine-lesioned rats

The present study reported the efficacy of antisense oligonucleotides specific for N-methyl-D-aspartate receptor one (NR1) in reduction of motor symptom in a rat parkinsonian model, the unilateral 6-hydroxydopamine-lesioned rat. Significant reductions in apomorphine-induced contralateral rotation were only seen in the NR1 antisense-treated lesioned rats (after a single intraparenchymal dose of antisense to the lesioned neostriatum; 15 nmol in 3 microl of saline) at 1 or 2 days after the treatment. No motor effect was seen in the lesioned animals with control treatments (sham, treatment using NR1 sense oligonucleotides, randomized oligonucleotides or saline, respectively). In contrast, significant increases in expression of NR1 mRNA in the lesioned neostriatum were seen in rats with control treatments but not in rats with NR1 antisense treatment. Importantly, in the lesioned neostriatum that was treated with NR1 antisense, a significant reduction in NR1 protein expression was found and NR1 immunoreactivity was seen to reduce in perikarya of presumed striatal medium spiny neurons. The present data indicate that a single dose of NR1 antisense ameliorates motor symptom in the rat model. The efficacy of NR1 antisense is likely to be mediated by a selective knockdown in expression of NR1 mRNA and proteins in the presumed medium spiny neurons.     

Increased sensitivity to N-methyl-D-aspartate receptor-mediated excitotoxicity in a mouse model of Huntington's disease

Previous work suggests N-methyl-D-aspartate receptor (NMDAR) activation may be involved in degeneration of medium-sized spiny striatal neurons in Huntington's disease (HD). Here we show that these neurons are more vulnerable to NMDAR-mediated death in a YAC transgenic FVB/N mouse model of HD expressing full-length mutant huntingtin, compared with wild-type FVB/N mice. Excitotoxic death of these neurons was increased after intrastriatal injection of quinolinate in vivo, and after NMDA but not AMPA exposure in culture. NMDA-induced cell death was abolished by an NR2B subtype-specific antagonist. In contrast, NMDAR-mediated death of cerebellar granule neurons was not enhanced, consistent with cell-type and NMDAR subtype specificity. Moreover, increased NMDA-evoked current amplitude and caspase-3 activity were observed in transgenic striatal neurons. Our data support a role for NR2B-subtype NMDAR activation as a trigger for selective neuronal degeneration in HD.     

Subcellular segregation of distinct heteromeric NMDA glutamate receptors in the striatum

Functional N-methyl-d-aspartate (NMDA) glutamate receptors are composed of heteromeric complexes of NR1, the obligatory subunit for channel activity, and NR2 or NR3 family members, which confer variability in the properties of the receptors. Recent studies have provided evidence for the existence of both binary (containing NR1 and either NR2A or NR2B) and ternary (containing NR1, NR2A, and NR2B) receptor complexes in the adult mammalian brain. However, the mechanisms regulating subunit assembly and receptor localization are not well understood. In the CNS, NMDA subunits are present both at intracellular sites and the post-synaptic membrane of neurons. Using biochemical protein fractionation and co-immunoprecipitation approaches we have found that in rat striatum binary NMDA receptors are widely distributed, and can be identified in the light membrane, synaptosomal membrane, and synaptic vesicle-enriched subcellular compartments. In contrast, ternary receptors are found exclusively in the synaptosomal membranes. When striatal proteins are chemically cross-linked prior to subcellular fractionation, ternary NMDA receptors can be precipitated from the light membrane and synaptic vesicle-enriched fractions where this type of receptor complex is not detectable under normal conditions. These findings suggest differential targeting of distinct types of NMDA receptor assemblies between intracellular and post-synaptic sites based on subunit composition. This targeting may underlie important differences in the regulation of the transport pathways involved in both normal as well as pathological receptor functions.     

Distinct aggregation and cell death patterns among different types of primary neurons induced by mutant huntingtin protein

Aggregation of disease proteins is believed to be a central event in the pathology of polyglutamine diseases, whereas the relationship between aggregation and neuronal death remains controversial. We investigated this question by expressing mutant huntingtin (htt) with a defective adenovirus in different types of neurons prepared from rat cerebral cortex, striatum or cerebellum. The distribution pattern of inclusions is not identical among different types of primary neurons. On day 2 after infection, cytoplasmic inclusions are dominant in cortical and striatal neurons, whereas at day 4 the ratio of nuclear inclusions overtakes that of cytoplasmic inclusions. Meanwhile, nuclear inclusions are always predominantly present in cerebellar neurons. The percentage of inclusion-positive cells is highest in cerebellar neurons, whereas mutant htt induces cell death most remarkably in cortical neurons. As our system uses htt exon 1 protein and thus aggregation occurs independently from cleavage of the full-length htt, our observations indicate that the aggregation process is distinct among different neurons. Most of the neurons containing intracellular (either nuclear or cytoplasmic) aggregates are viable. Our findings suggest that the process of mutant htt aggregation rather than the resulting inclusion body is critical for neuronal cell death.     

HIV-1 Tat through phosphorylation of NMDA receptors potentiates glutamate excitotoxicity

Toxic effects of HIV-1 proteins contribute to altered function and decreased survival of select populations of neurons in HIV-1-infected brain. One such HIV-1 protein, Tat, can activate calcium release from IP3-sensitive intracellular pools, induce calcium influx in neural cells, and, as a result, can increase neuronal cell death. Here, we provide evidence that Tat potentiates excitatory amino acid (glutamate and NMDA) triggered calcium flux, as well as glutamate- and staurosporine-mediated neurotoxicity. Calcium flux in cultured rat hippocampal neurons triggered by the transient application of glutamate or NMDA was facilitated by pre-exposure to Tat. Facilitation of glutamate-triggered calcium flux by Tat was prevented by inhibitors of ADP-ribosylation of G(i)/G(o) proteins (pertussis toxin), protein kinase C (H7 and bisindolymide), and IP3-mediated calcium release (xestospongin C), but was not prevented by an activator of G(s) (cholera toxin) or an inhibitor of protein kinase A (H89). Facilitation of NMDA-triggered calcium flux by Tat was reversed by inhibitors of tyrosine kinase (genestein and herbimycin A) and by an inhibitor of NMDA receptor function (zinc). Tat increased 32P incorporation into NMDA receptor subunits NR2A and NR2B and this effect was blocked by genestein. Subtoxic concentrations of Tat combined with subtoxic concentrations of glutamate or staurosporine increased neuronal cell death significantly. Together, these findings suggest that NMDA receptors play an important role in Tat neurotoxicity and the mechanisms identified may provide additional therapeutic targets for the treatment of HIV-1 associated dementia.     

Modulation of D2R-NR2B interactions in response to cocaine

Dopamine-glutamate interactions in the neostriatum determine psychostimulant action, but the underlying molecular mechanisms remain elusive. Here we found that dopamine stimulation by cocaine enhances a heteroreceptor complex formation between dopamine D2 receptors (D2R) and NMDA receptor NR2B subunits in the neostriatum in vivo. The D2R-NR2B interaction is direct and occurs in the confined postsynaptic density microdomain of excitatory synapses. The enhanced D2R-NR2B interaction disrupts the association of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) with NR2B, reduces NR2B phosphorylation at a CaMKII-sensitive site (Ser1303), and inhibits NMDA receptor-mediated currents in medium-sized striatal neurons. Furthermore, the regulated D2R-NR2B interaction is critical for constructing behavioral responsiveness to cocaine. Our findings here uncover a direct and dynamic D2R-NR2B interaction in striatal neurons in vivo. This type of dopamine-glutamate integration at the receptor level may be responsible for synergistically inhibiting the D2R-mediated circuits in the basal ganglia and fulfilling the stimulative effect of psychostimulants.     

Immunolesioning of Glutamate Receptor GluR1-Containing Neurons in the Rat Neostriatum Using a Novel Immunotoxin

1. To investigate the potency of a novel immunotoxin that is specific for glutamate receptor GluR1, a subunit of the -amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-type receptor channel, immunolesioning was performed.2. A ribosome-inactivating protein, trichosanthin (TCS), was isolated and conjugated to the goat anti-rabbit IgG antibody molecule. The anti-rabbit antibody–TCS complex was preincubated with GluR1-specific rabbit antibody to produce a GluR1-specific immunotoxin. The immunotoxin was unilaterally administered into either the neostriatum or the lateral ventricle of rats.3. Immunoreactivity for GluR1 or GluR4 was revealed in perfuse-fixed sections of the neostriatum obtained from the lesioned and control animals by immunocytochemistry. After ventricular or striatal injections of the immunotoxin, depletions of GluR1-immunoreactive neurons, the presumed GABAergic interneurons in the neostriatum, were found. Depletions of GluR4-immunoreactive perikarya, the presumed same subpopulation of striatal interneurons, were also found. In addition, no change in the pattern of distribution of immunoreactivity for GluR2 or glial fibrillary acidic protein was found in the lesioned neostriatum. These results indicate that the novel GluR1 immunotoxin is potent and specific.4. In addition, striatal application of the immunotoxin caused a greater depletion in the number of GluR1-immunoreactive neurons. The present results also indicate that the route of immunotoxin application may be important in producing specific lesions.     

Neural cell adhesion molecule-associated polysialic acid inhibits NR2B-containing N-methyl-D-aspartate receptors and prevents glutamate-induced cell death

The neural cell adhesion molecule (NCAM) and its associated glycan polysialic acid play important roles in the development of the nervous system and N-methyl-D-aspartate(NMDA)receptor-dependent synaptic plasticity in the adult. Here, we investigated the influence of polysialic acid on NMDA receptor activity. We found that glutamate-elicited NMDA receptor currents in cultured hippocampal neurons were reduced by approximately 30% with the application of polysialic acid or polysialylated NCAM but not by the sialic acid monomer, chondroitin sulfate, or non-polysialylated NCAM. Polysialic acid inhibited NMDA receptor currents elicited by 3 microm glutamate but not by 30 microm glutamate, suggesting that polysialic acid acts as a competitive antagonist, possibly at the glutamate binding site. The polysialic acid induced effects were mimicked and fully occluded by the NR2B subunit specific antagonist, ifenprodil. Recordings from single synaptosomal NMDA receptors reconstituted in lipid bilayers revealed that polysialic acid reduced open probability but not the conductance of NR2B-containing NMDA receptors in a polysialic acid and glutamate concentration-dependent manner. The activity of single NR2B-lacking synaptosomal NMDA receptors was not affected by polysialic acid. Application of polysialic acid to hippocampal cultures reduced excitotoxic cell death induced by low micromolar concentration of glutamate via activation of NR2B-containing NMDA receptors, whereas enzymatic removal of polysialic acid resulted in increased cell death that occluded glutamate-induced excitotoxicity. These observations indicate that the cell adhesion molecule-associated glycan polysialic acid is able to prevent excitotoxicity via inhibition of NR2B subunit-containing NMDA receptors.     

Differential involvement of cell cycle reactivation between striatal and cortical neurons in cell death induced by 3-nitropropionic acid

Recent evidence suggests that unscheduled cell cycle activity leads to neuronal cell death. 3-Nitropropionic acid (3-NP) is an irreversible inhibitor of succinate dehydrogenase and induces cell death in both striatum and cerebral cortex. Here we analyzed the involvement of aberrant cell cycle progression in 3-NP-induced cell death in these brain regions. 3-NP reduced the level of cyclin-dependent kinase inhibitor p27 in striatum but not in cerebral cortex. 3-NP also induced phosphorylation of retinoblastoma protein, a marker of cell cycle progression at late G(1) phase, only in striatum. Pharmacological experiments revealed that cyclin-dependent kinase activity and N-methyl-d-aspartate (NMDA) receptor were cooperatively involved in cell death by 3-NP in striatal neurons, whereas only NMDA receptor was involved in 3-NP-induced neurotoxicity in cortical neurons. Death of striatal neurons was preceded by elevation of somatic Ca(2+) and activation of calpain, a Ca(2+)-dependent protease. Both striatal p27 down-regulation and cell death provoked by 3-NP were dependent on calpain activity. Moreover, transfection of p27 small interfering RNA reduced striatal cell viability. In cortical neurons, however, there was no change in somatic Ca(2+) and calpain activity by 3-NP, and calpain inhibitors were not protective. These results suggest that 3-NP induces aberrant cell cycle progression and neuronal cell death via p27 down-regulation by calpain in striatum but not in the cerebral cortex. This is the first report for differential involvement of cell cycle reactivation in different brain regions and lightens the mechanism for region-selective vulnerability in human disease, including Huntington disease.     

Different capacities of various NMDA receptor antagonists to prevent ischemia-induced neurodegeneration in human cultured NT2 neurons

In the present study, human NT2 neurons obtained from embryonic teratocarcinoma (NT2) cells were established as human in-vitro model to investigate the mechanisms associated with hypoxia/ischemia-induced neuronal injury. NT2 neurons express functional NMDA receptors that are of particular significance for hypoxia/ischemia-related neuronal damage. In patch-clamp recordings under normoxic conditions, NMDA (plus 10 microM glycine)-induced inward currents (EC(50)=43.7 microM) were distinctly antagonized by memantine, a blocker of the receptor channel, but only slightly by 5,7-dichlorokynurenic acid (DCKA), a glycine(B) binding site antagonist. Immunohistochemistry demonstrated that the NT2 neurons are mostly GABAergic; they predominantly express the NMDA receptor subunits NR2B and NR2C, and lower levels of NR1 and, particularly, of NR2A. Upon glucose and oxygen deprivation for 3h the loss of cell viability measured directly after 3h was higher than after application of either hypoxia or aglycemia as assessed by propidium iodide flow cytometry. Ischemic conditions significantly reduced the NMDA responses associated with a decrease in EC(50) and decreased mitochondrial membrane potential as detected by JC-1 flow cytometry. Memantine (50 microM) and CGS19755 (a competitive NMDA receptor antagonist; 10 microM) reduced ischemia-induced cell death, in contrast to DCKA (10 microM). In conclusion, in the present human in-vitro model for studying the molecular mechanisms associated with ischemic injury, neuroprotection could be achieved with NMDA receptor antagonists but not with a glycine(B) binding site antagonist. Accordingly, glycine antagonists might not represent an optimal therapeutic strategy for preventing ischemic neuronal damage in contrast to NMDA receptor antagonists like memantine.     

Fyn is required for haloperidol-induced catalepsy in mice

Fyn-mediated tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunits has been implicated in various brain functions, including ethanol tolerance, learning, and seizure susceptibility. In this study, we explored the role of Fyn in haloperidol-induced catalepsy, an animal model of the extrapyramidal side effects of antipsychotics. Haloperidol induced catalepsy and muscle rigidity in the control mice, but these responses were significantly reduced in Fyn-deficient mice. Expression of the striatal dopamine D(2) receptor, the main site of haloperidol action, did not differ between the two genotypes. Fyn activation and enhanced tyrosine phosphorylation of the NMDA receptor NR2B subunit, as measured by Western blotting, were induced after haloperidol injection of the control mice, but both responses were significantly reduced in Fyn-deficient mice. Dopamine D(2) receptor blockade was shown to increase both NR2B phosphorylation and the NMDA-induced calcium responses in control cultured striatal neurons but not in Fyn-deficient neurons. Based on these findings, we proposed a new molecular mechanism underlying haloperidol-induced catalepsy, in which the dopamine D(2) receptor antagonist induces striatal Fyn activation and the subsequent tyrosine phosphorylation of NR2B alters striatal neuronal activity, thereby inducing the behavioral changes that are manifested as a cataleptic response.     

Subtype-specific enhancement of NMDA receptor currents by mutant huntingtin

Evidence suggests that NMDA receptor-mediated neurotoxicity plays a role in the selective neurodegeneration underlying Huntington's disease (HD). The gene mutation that causes HD encodes an expanded polyglutamine tract of >35 in huntingtin, a protein of unknown function. Both huntingtin and NMDA receptors interact with cytoskeletal proteins, and, for NMDA receptors, such interactions regulate surface expression and channel activity. To determine whether mutant huntingtin alters NMDA receptor expression or function, we coexpressed mutant or normal huntingtin, containing 138 or 15 glutamine repeats, respectively, with NMDA receptors in a cell line and then assessed receptor channel function by patch-clamp recording and surface expression by western blot analysis. It is interesting that receptors composed of NR1 and NR2B subunits exhibited significantly larger currents when coexpressed with mutant compared with normal huntingtin. Moreover, this effect was selective for NR1/NR2B, as NR1/NR2A showed similar currents when coexpressed with mutant versus normal huntingtin. However, ion channel properties and total surface expression of the NR1 subunit were unchanged in cells cotransfected with NR1/NR2B and mutant huntingtin. Our results suggest that mutant huntingtin may increase numbers of functional NR1/NR2B-type receptors at the cell surface. Because NR1/NR2B is the predominant NMDA receptor subtype expressed in medium spiny neostriatal neurons, our findings may help explain the selective vulnerability of these neurons in HD.     

N-methyl-D-aspartate receptor subtype mediated bidirectional control of p38 mitogen-activated protein kinase

N-methyl-d-aspartate receptor (NMDAR) stimulation activates many downstream mechanisms involved in both cell survival and cell death. The manner in which the NMDAR regulates one of these pathways, the p38 mitogen-activated protein kinase (p38) pathway, is currently unknown. In the present study, we have defined a developmental-, concentration-, and time-dependent phosphorylation and subsequent dephosphorylation of p38. In cultured hippocampal neurons 7-8 days in vitro (DIV7-8), NMDAR stimulation leads to a concentration-dependent increase in p38 phosphorylation (phospho-p38). However, in more mature neurons (>DIV17) application of NMDA produces concentration-dependent effects, such that low concentrations result in sustained increases in phospho-p38 levels, and high concentrations dephosphorylate p38 within 5 min. Conantokin G, an antagonist of NR1/2A/2B and NR1/2B receptors, inhibits p38 phosphorylation, while NR1/2B-specific antagonists prevent the rapid dephosphorylation of p38 without affecting p38 activation. Furthermore, inhibition of calcineurin prevents the activation of p38, whereas inhibition of phosphoinositide 3-kinase (PI3K) prevents the rapid dephosphorylation of p38. Our results support the presence of subtype-dependent pathways regulating p38 activation and deactivation: one involves NR1/2A/2B receptors activating calcineurin and resulting in p38 phosphorylation, and the other utilizes NR1/2B receptors binding to and activating PI3K and leading to the dephosphorylation of p38 in a manner involving both NR1/2A/2B receptor activation and tyrosine phosphorylation of NR2B. The ability of NMDAR subtype-specific mechanisms to regulate p38 has implications for NMDAR-mediated synaptic plasticity, gene regulation, and excitotoxicity.     

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.     

Chronic ethanol exposure delays the 'developmental switch' of the NMDA receptor 2A and 2B subunits in cultured cerebellar granule neurons

Chronic ethanol treatment of cultured neurons from various brain areas has been found to increase NMDA receptor function and to alter the levels of some NMDA receptor subunit proteins. Because the cultured neurons are exposed to ethanol during a period when the NMDA receptor is undergoing developmental changes in subunit expression, we wished to determine whether ethanol treatment alters this developmental pattern. We found that 3 days of treatment of cerebellar granule neurons with ethanol, which was previously reported to increase NMDA receptor function, resulted in a delay in the 'developmental switch' of the NR2A and NR2B subunits, i.e. the developmental decrease in NR2B and increase in NR2A protein expression. As a result, the level of NR2B was higher, and that of NR2A was lower, in the ethanol-treated cells than in control cells. Cross-linking experiments showed that the changes in total receptor subunit proteins levels were reflected in cell-surface expressed proteins, indicating changes in the amount of functional receptors. These results were confirmed by a higher potency of glycine at the NMDA receptor in the ethanol-treated cells, as determined by NMDA/glycine-induced increases in intracellular Ca(2+). The results suggest that the mechanism by which ethanol alters NMDA receptor expression in cultured neurons, where receptors are undergoing development, differs from the mechanism of ethanol's effect on NMDA receptors in adult brain. Changes in the proportion of NR2A and NR2B subunits may contribute to effects of ethanol on neuronal development.     

Transient Ischemia Differentially Increases Tyrosine Phosphorylation of NMDA Receptor Subunits 2A and 2B

Abstract: Activation of the N -methyl- d -aspartate (NMDA) receptor has been implicated in the events leading to ischemia-induced neuronal cell death. Recent studies have indicated that the properties of the NMDA receptor channel may be regulated by tyrosine phosphorylation. We have therefore examined the effects of transient cerebral ischemia on the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B in different regions of the rat brain. Transient (15 min) global ischemia was produced by the four-vessel occlusion procedure. The tyrosine phosphorylation of NR2A and NR2B subunits was examined by immunoprecipitation with anti-tyrosine phosphate antibodies followed by immunoblotting with antibodies specific for NR2A or NR2B, and by immunoprecipitation with subunit-specific antibodies followed by immunoblotting with anti-phosphotyrosine antibodies. Transient ischemia followed by reperfusion induced large (23–29-fold relative to sham-operated controls), rapid (within 15 min of reperfusion), and sustained (for at least 24 h) increases in the tyrosine phosphorylation of NR2A and smaller increases in that of NR2B in the hippocampus. Ischemia-induced tyrosine phosphorylation of NR2 subunits in the hippocampus was higher than that of cortical and striatal NR2 subunits. The enhanced tyrosine phosphorylation of NR2A or NR2B may contribute to alterations in NMDA receptor function or in signaling pathways in the postischemic brain and may be related to pathogenic events leading to neuronal death.