Rescue of abnormal phenotypes of the delta2 glutamate receptor-null mice by mutant delta2 transgenes

The delta2 glutamate receptor (GluRdelta2) has a crucial role in cerebellar functions; disruption of GluRdelta2 alleles in mice (delta2(-/-)) impairs synapse formation and long-term depression, which is thought to underlie motor learning in the cerebellum, and consequently leads to motor discoordination. However, it has been unclear whether GluRdelta2 is activated by glutamate analogues. Here we introduced a GluRdelta2 transgene, which had a mutation (Arg514Lys) in the putative ligand-binding motif conserved in all mammalian ionotropic glutamate receptors (iGluRs) and their ancestral bacterial periplasmic amino-acid-binding proteins, into delta2(-/-) mice. Surprisingly, a mutant GluRdelta2 transgene, as well as a wild-type GluRdelta2 transgene, rescued all abnormal phenotypes of delta2(-/-) mice. Therefore, these results indicate that the conserved arginine residue, which is crucial for the binding of iGluRs to glutamate analogues, is not essential for the restoration of GluRdelta2 functions in delta2(-/-) mice.     

Binding of glutamate receptor delta2 to its scaffold protein, Delphilin, is regulated by PKA

The glutamate receptor delta2 (GluRdelta2) is selectively expressed in cerebellar Purkinje cells and plays an important role in motor learning, motor coordination, and long-term depression. Delphilin is identified as a GluRdelta2-interacting protein, selectively expressed in Purkinje cell-parallel fiber synapses, and specifically interacts with the GluRdelta2 C-terminus via its PDZ domain. Here, surface plasmon resonance analyses showed that Delphilin PDZ bound to GluRdelta2 C-terminal peptide (DPDRGTSI), but not to its phosphopeptides (DPDRGphosphoTSI and DPDRGTphosphoSI). We showed the incorporation of phosphate into threonine at -2 (-2T) and serine at -1 (-1S) of GluRdelta2 C-terminus by cAMP-dependent protein kinase (PKA) in vitro. In the experiments using heterologous expression system, Delphilin coimmunoprecipitated with GluRdelta2 was dramatically decreased under the condition with forskolin and isobutylmethylxanthine, which led to cAMP-dependent phosphorylation by PKA. Thus, phosphorylation of -2T and/or -1S of GluRdelta2 C-terminus by PKA may regulate the binding of GluRdelta2 to its scaffolding protein, Delphilin.     

A new motif necessary and sufficient for stable localization of the delta2 glutamate receptors at postsynaptic spines

The number of each subclass of ionotropic glutamate receptors (iGluRs) at the spines is differentially regulated either constitutively or in a neuronal activity-dependent manner. The delta2 glutamate receptor (GluRdelta2) is abundantly expressed at the spines of Purkinje cell dendrites and controls synaptic plasticity in the cerebellum. To obtain clues to the trafficking mechanism of the iGluRs, we expressed wild-type or mutant GluRdelta2 in cultured hippocampal and Purkinje neurons and analyzed their intracellular localization using immunocytochemical techniques. Quantitative analysis revealed that deletion of the 20 amino acids at the center of the C terminus (region E) significantly reduced the amount of GluRdelta2 protein at the spines in both types of neurons. This effect was partially antagonized by the inhibition of endocytosis by high dose sucrose treatment or coexpression of dominant negative dynamin. In addition, mutant GluRdelta2 lacking the E region (GluRdelta2DeltaE), but not wild-type GluRdelta2, was found to colocalize with the endosomal markers Rab4 and Rab7. Moreover, the antibody-feeding assay revealed that GluRdelta2DeltaE was internalized more rapidly than GluRdelta2wt. These results indicate that the E region (more specifically, a 12-amino-acid-long segment of the E2 region) is necessary for rendering GluRdelta2 resistant to endocytosis from the cell surface at the spines. Furthermore, insertion of the E2 region alone into the C terminus of the GluR1 subtype of iGluRs was sufficient to increase the amount of GluR1 proteins in the spines. Therefore, we propose that the E2 region of GluRdelta2 is necessary, and also sufficient, to inhibit endocytosis of the receptor from postsynaptic membranes.     

Characterization of the delta2 glutamate receptor-binding protein delphilin: Splicing variants with differential palmitoylation and an additional PDZ domain

The glutamate receptor delta2 (GluRdelta2) is predominantly expressed at parallel fiber-Purkinje cell postsynapses and plays crucial roles in synaptogenesis and synaptic plasticity. Although the mechanism by which GluRdelta2 functions remains unclear, its lack of channel activity and its role in controlling the endocytosis of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors have suggested that GluRdelta2 may convey signals by interacting with intracellular signaling molecules. Among several proteins that interact with GluRdelta2, delphilin is unique in that it is selectively expressed at parallel fiber-Purkinje cell synapses and that, in addition to a single PDZ domain, it contains a formin homology domain that is thought to regulate actin dynamics. Here, we report a new isoform of delphilin, designated as L-delphilin, that has alternatively spliced N-terminal exons encoding an additional PDZ domain. Although original delphilin, designated S-delphilin, was palmitoylated at the N terminus, this region was spliced out in L-delphilin. As a result, S-delphilin was associated with plasma membranes in COS cells and dendritic spines in hippocampal neurons, whereas L-delphilin formed clusters in soma and dendritic shafts. In addition, S-delphilin, but not L-delphilin, facilitated the expression of GluRdelta2 on the cell surface. These results indicate that, like PSD-95 and GRIP/ABP, delphilin isoforms with differential palmitoylation and clustering capabilities may provide two separate intracellular and surface GluRdelta2 pools and may control GluRdelta2 signaling in Purkinje cells.     

Cerebellar regulation mechanisms learned from studies on GluRdelta2

The amino acid sequence suggests that glutamate receptor delta2 (GluRdelta2) belongs to an ionotropic GluR (iGluR) subunit family. However, neither the direct binding to glutamate nor the incorporation into any native iGluRs has been demonstrated. One prominent feature of GluRdelta2 is its predominant expression at parallel fiber-Purkinje cell synapses in the cerebellum. Knockdown or knockout of GluRdelta2 impairs synaptic plasticity, stabilization, elimination, motor control, and learning. Therefore, GluRdelta2 plays a crucial role in the cerebellar function. Several ataxic spontaneous mutant mice have defects in the GluRdelta gene. Numerous proteins interacting with GluRdelta2 have been identified. Recent in vivo studies on GluRdelta2 knockout mice shed light on the mechanism by which GluRdelta2 deficiency causes ataxia and unveiled some secondary influence of the GluRdelta2 deficiency on the function of the central nervous system. Studies on GluRdelta2 might provide unique clues regarding not only the molecular mechanism of synaptic regulations but also the functioning mechanism of the entire cerebellar system.     

Oxidative stress, nitric oxide, and the mechanisms of cell death in Lurcher Purkinje cells

Oxidative stress is postulated to play a role in cell death in many neurodegenerative diseases. As a model of neonatal neuronal cell death, we have examined the role of oxidative stress in Purkinje cell death in the heterozygous Lurcher mutant (+/Lc). Lurcher is a gain of function mutation in the delta2 glutamate receptor (GluRdelta2) that turns the receptor into a leaky membrane channel, resulting in chronic depolarization of +/Lc Purkinje cells starting around the first week of postnatal development. Virtually, all +/Lc Purkinje cells die by the end of the first postnatal month. To investigate the role of oxidative stress in +/Lc Purkinje cell death, we have examined nitric oxide synthase (NOS) activity and the expression of two markers for oxidative stress, nitrotyrosine and manganese super oxide dismutase (MnSOD), in wild type and +/Lc Purkinje cells at P10, P15, and P25. The results show that NOS activity and immunolabeling for nitrotyrosine and MnSOD are increased in +/Lc Purkinje cells. To determine whether peroxynitrite formation is a prerequisite for +/Lc Purkinje cell death, +/Lc mutants were crossed with an alpha-nNOS knockout mutant (nNOSalpha(-/-)) to reduce the production of NO. Analysis of the double mutants showed that blocking alpha-nNOS expression does not rescue +/Lc Purkinje cells. However, we present evidence for sustained NOS activity and nitrotyrosine formation in the GluRdelta2(+/Lc):nNOS(-/-) double mutant Purkinje cells, which suggests that the failure to rescue GluRdelta2(+/Lc):nNOS(-/-) Purkinje cells may be explained by the induction of alternative nNOS isoforms.     

Orphan glutamate receptor delta1 subunit required for high-frequency hearing

The function of the orphan glutamate receptor delta subunits (GluRdelta1 and GluRdelta2) remains unclear. GluRdelta2 is expressed exclusively in the Purkinje cells of the cerebellum, and GluRdelta1 is prominently expressed in inner ear hair cells and neurons of the hippocampus. We found that mice lacking the GluRdelta1 protein displayed significant cochlear threshold shifts for frequencies of >16 kHz. These deficits correlated with a substantial loss of type IV spiral ligament fibrocytes and a significant reduction of endolymphatic potential in high-frequency cochlear regions. Vulnerability to acoustic injury was significantly enhanced; however, the efferent innervation of hair cells and the classic efferent inhibition of outer hair cells were unaffected. Hippocampal and vestibular morphology and function were normal. Our findings show that the orphan GluRdelta1 plays an essential role in high-frequency hearing and ionic homeostasis in the basal cochlea, and the locus encoding GluRdelta1 represents a candidate gene for congenital or acquired high-frequency hearing loss in humans.     

The Lurcher mutation of an alpha-amino-3-hydroxy-5-methyl- 4-isoxazolepropionic acid receptor subunit enhances potency of glutamate and converts an antagonist to an agonist

A point mutation of the GluRdelta2 (A654T) glutamate receptor subunit converts it into a functional channel, and a spontaneous mutation at this site is thought to be responsible for the neurodegeneration of neurons in the Lurcher mouse. This mutation is located in a hydrophobic region of the M3 domain of this subunit, and this alanine is conserved throughout many of the glutamate receptors. We show here that site-directed mutagenesis of the homologous alanine (A636T; GluR1-L(c)) in the GluR1 AMPA receptor subunit alters its channel properties. The apparent potencies of both kainate and glutamate were increased 85- and 2000-fold, respectively. Furthermore, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)was converted from a competitive antagonist into a potent agonist. Our results demonstrate that a single amino acid within or near the putative second transmembrane region of the GluR1 subunit is critical for the binding/gating properties of this AMPA receptor.     

Roles of diverse glutamate receptors in brain functions elucidated by subunit-specific and region-specific gene targeting

Glutamate receptor (GluR) channels play a major role in fast excitatory synaptic transmission in vertebrate central nervous system. We revealed the molecular diversity of the GluR channel by molecular cloning and investigated their physiological roles by subunit-specific gene targeting. NMDA receptor GluRepsilon1 KO mice showed increase in thresholds for hippocampal long-term potentiation and hippocampus-dependent contextual learning. The mutant mice performed delay eyeblink conditioning, but failed to learn trace eyeblink conditioning. GluRepsilon1 mutant suffered less brain injury after focal cerebral ischemia. NMDA receptor GluRepsilon2 KO mice showed impairment of the whisker-related neural pattern formation and suckling response, and died shortly after birth. Heterozygous (+/-) GluRepsilon2 mutant mice were viable and showed enhanced startle response to acoustic stimuli. GluRdelta2, a member of novel GluR channel subfamily we found by molecular cloning, is selectively expressed in the Purkinje cells of the cerebellum. GluRdelta2 KO mice showed impairments of cerebellar synaptic plasticity and synapse stability. GluRdelta2 KO mice exhibited impairment in delay eyeblink conditioning, but learned normally trace eyeblink conditioning. The phenotypes of NMDA receptor subunits and GluRdelta2 mutant mice suggest that diverse GluR subunits play differential roles in the brain functions.     

Expression of zebrafish glutamate receptor delta2 in neurons with cerebellum-like wiring

Mammalian glutamate receptor (GluR) delta2 is selectively expressed in cerebellar Purkinje cells and plays key roles in cerebellar plasticity, motor learning, and neural wiring. Here, we isolated cDNA encoding the zebrafish ortholog of mammalian GluRdelta2. We found that in adult zebrafish brain, glurdelta2 mRNA was expressed not only in cerebellar Purkinje cells, but also in the crest cells of the medial octavolateral nucleus (MON) and the type I neurons of the optic tectum. Immunohistochemical analysis revealed that zebrafish GluRdelta2 proteins were selectively localized in the apical dendrites of these neurons. Interestingly, the crest cells of the MON and the type I neurons of the optic tectum receive large numbers of parallel fiber inputs at the apical dendrites and sensory inputs at the proximal or basal dendrites. These results suggest that the expression of zebrafish GluRdelta2 is selective for cerebellum-like neural wiring with large numbers of parallel fiber inputs.     

The protein-tyrosine phosphatase PTPMEG interacts with glutamate receptor delta 2 and epsilon subunits

Glutamate receptor (GluR) delta2 is selectively expressed in cerebellar Purkinje cells and plays a crucial role in cerebellum-dependent motor learning. Although GluRdelta2 belongs to an ionotropic GluR family, little is known about its pharmacological features and downstream signaling cascade. To study molecular mechanisms underlying GluRdelta2-dependent motor learning, we employed yeast two-hybrid screening to isolate GluRdelta2-interacting molecules and identified protein-tyrosine phosphatase PTPMEG. PTPMEG is a family member of band 4.1 domain-containing protein-tyrosine phosphatases and is expressed prominently in brain. Here, we showed by in situ hybridization analysis that the PTPMEG mRNA was enriched in mouse thalamus and Purkinje cells. We also showed that PTPMEG interacted with GluRdelta2 as well as with N-methyl-d-aspartate receptor GluRepsilon1 in cultured cells and in brain. PTPMEG bound to the putative C-terminal PDZ target sequence of GluRdelta2 and GluRepsilon1 via its PDZ domain. Examination of the effect of PTPMEG on tyrosine phosphorylation of GluRepsilon1 unexpectedly revealed that PTPMEG enhanced Fyn-mediated tyrosine phosphorylation of GluRepsilon1 in its PTPase activity-dependent manner. Thus, we conclude that PTPMEG associates directly with GluRdelta2 and GluRepsilon1. Moreover, our data suggest that PTPMEG plays a role in signaling downstream of the GluRs and/or in regulation of their activities through tyrosine dephosphorylation.     

A novel protein complex linking the delta 2 glutamate receptor and autophagy: implications for neurodegeneration in lurcher mice

Autophagy is a pathway for bulk degradation of subcellular constituents that is hyperactivated in many neurodegenerative conditions. It has been considered a second form of programmed cell death. Death of cerebellar Purkinje cells in lurcher animals is due to a mutation in GluRdelta2 that results in its constitutive activation. Here we have identified protein interactions between GluRdelta2, a novel isoform of a PDZ domain-containing protein (nPIST) that binds to this receptor, and Beclin1. nPIST and Beclin1 can synergize to induce autophagy. GluRdelta2(Lc), but not GluRdelta2(wt), can also induce autophagy. Furthermore, dying lurcher Purkinje cells contain morphological hallmarks of autophagic death in vivo. These results provide strong evidence that a direct link exists between GluRdelta2(Lc) receptor and stimulation of the autophagic pathway in dying lurcher Purkinje cells.     

Enhancement of both long-term depression induction and optokinetic response adaptation in mice lacking delphilin

In the cerebellum, Delphilin is expressed selectively in Purkinje cells (PCs) and is localized exclusively at parallel fiber (PF) synapses, where it interacts with glutamate receptor (GluR) delta2 that is essential for long-term depression (LTD), motor learning and cerebellar wiring. Delphilin ablation exerted little effect on the synaptic localization of GluRdelta2. There were no detectable abnormalities in cerebellar histology, PC cytology and PC synapse formation in contrast to GluRdelta2 mutant mice. However, LTD induction was facilitated at PF-PC synapses in Delphilin mutant mice. Intracellular Ca(2+) required for the induction of LTD appeared to be reduced in the mutant mice, while Ca(2+) influx through voltage-gated Ca(2+) channels and metabotropic GluR1-mediated slow synaptic response were similar between wild-type and mutant mice. We further showed that the gain-increase adaptation of the optokinetic response (OKR) was enhanced in the mutant mice. These findings are compatible with the idea that LTD induction at PF-PC synapses is a crucial rate-limiting step in OKR gain-increase adaptation, a simple form of motor learning. As exemplified in this study, enhancing synaptic plasticity at a specific synaptic site of a neural network is a useful approach to understanding the roles of multiple plasticity mechanisms at various cerebellar synapses in motor control and learning.     

Rapid activation, desensitization, and resensitization of synaptic channels of crayfish muscle after glutamate pulses.

Completely desensitizing excitatory channels were activated in outside-out patches of crayfish muscle membrane by applying glutamate pulses with switching times of approximately 0.2 ms for concentration changes. Channels were almost completely activated with 10 mM glutamate. Maximum activation was reached within 0.4 ms with greater than or equal to 1 mM glutamate. Channel open probability decayed with a time constant of desensitization of 2 ms with 10 mM glutamate and more rapidly at lower glutamate concentrations. The rate of beginnings of bursts (average number of beginnings of bursts per time bin) decayed even faster but approximately in proportion to the glutamate concentration. The dose-response curve for the channel open probability and for the rate of bursts had a maximum double-logarithmic slope of 5.1 and 4.2, respectively. Channels desensitized completely without opening at very low or slowly rising glutamate concentrations. Desensitization thus originates from a closed channel state. Resensitization was tested by pairs of completely desensitizing glutamate pulses. Sensitivity to the second pulse returned rapidly at pulse intervals between 1 and 2 ms and was almost complete with an interval of 3 ms. Schemes of channel activation by up to five glutamate binding steps, with desensitization by glutamate binding from closed states, are discussed. At high agonist concentrations bursts are predominantly terminated by desensitization. Quantal currents are generated by pulses of greater than 1 mM glutamate, and their decay is determined by the duration of presence of glutamate and possibly by desensitization.     

Purkinje cell-specific and inducible gene recombination system generated from C57BL/6 mouse ES cells

Spatiotemporally restricted gene targeting is needed for analyzing the functions of various molecules in a variety of biological phenomena. We have generated an inducible cerebellar Purkinje cell-specific gene targeting system. This was achieved by establishing a mutant mouse line (D2CPR) from a C57BL/6 mouse ES cell line, which expressed a fusion protein consisting of the Cre recombinase and the progesterone receptor (CrePR). The Purkinje cell-specific expression of CrePR was attained by inserting CrePR into the glutamate receptor delta2 subunit (GluRdelta2) gene, which was expressed specifically in the Purkinje cells. Using the transgenic mice carrying the Cre-mediated reporter gene, we showed that the antiprogesterone RU486 could induce recombinase activity of the CrePR protein specifically in the mature cerebellar Purkinje cells of the D2CPR line. Thus this mutant line will be a useful tool for studying the molecular function of mature Purkinje cells by manipulating gene expression in a temporally restricted manner.     

Calcium-dependent,slow desensitization distinguishes different types of glutamate receptors

1. L-Glutamate, the most likely transmitter of rapid excitatory synaptic interactions in the brain and spinal cord, is a potent neurotoxin. Mechanisms that terminate the action of glutamate are, therefore, likely to be important for maintaining the integrity of glutaminoceptive neurons. In this study, we show that glutamate currents evoked in voltage-clamped chick motoneurons fade during prolonged or repeated application of glutamate by pressure ejection from nearby pipettes. 2. The magnitude of the decline depends on the Ca2+/Mg2+ ratio in the extracellular medium. With Ca2+ = 10.0 mM and no added Mg, the steady-state glutamate current amounted to 50% of the initial value. 3. Single-channel measurements indicate that the fade is due to receptor desensitization rather than to agonist-induced channel blockade, as the mean channel open time within bursts is independent of the agonist concentration. 4. Application of more selective agonists showed that Ca2+-dependent slow desensitization involved only G1 (NMDA) receptors. G2 responses (activated by kainate and quisqualate) did not exhibit this slow phase of desensitization under the same conditions.     

A point mutation in the pore region alters gating, Ca(2+) blockage, and permeation of olfactory cyclic nucleotide-gated channels

Upon stimulation by odorants, Ca(2+) and Na(+) enter the cilia of olfactory sensory neurons through channels directly gated by cAMP. Cyclic nucleotide-gated channels have been found in a variety of cells and extensively investigated in the past few years. Glutamate residues at position 363 of the alpha subunit of the bovine retinal rod channel have previously been shown to constitute a cation-binding site important for blockage by external divalent cations and to control single-channel properties. It has therefore been assumed, but not proven, that glutamate residues at the corresponding position of the other cyclic nucleotide-gated channels play a similar role. We studied the corresponding glutamate (E340) of the alpha subunit of the bovine olfactory channel to determine its role in channel gating and in permeation and blockage by Ca(2+) and Mg(2+). E340 was mutated into either an aspartate, glycine, glutamine, or asparagine residue and properties of mutant channels expressed in Xenopus laevis oocytes were measured in excised patches. By single-channel recordings, we demonstrated that the open probabilities in the presence of cGMP or cAMP were decreased by the mutations, with a larger decrease observed on gating by cAMP. Moreover, we observed that the mutant E340N presented two conductance levels. We found that both external Ca(2+) and Mg(2+) powerfully blocked the current in wild-type and E340D mutants, whereas their blockage efficacy was drastically reduced when the glutamate charge was neutralized. The inward current carried by external Ca(2+) relative to Na(+) was larger in the E340G mutant compared with wild-type channels. In conclusion, we have confirmed that the residue at position E340 of the bovine olfactory CNG channel is in the pore region, controls permeation and blockage by external Ca(2+) and Mg(2+), and affects channel gating by cAMP more than by cGMP.     

Cbln1 is essential for interaction-dependent secretion of Cbln3

Cbln1 and the orphan glutamate receptor GluRdelta2 are pre- and postsynaptic components, respectively, of a novel transneuronal signaling pathway regulating synapse structure and function. We show here that Cbln1 is secreted from cerebellar granule cells in complex with a related protein, Cbln3. However, cbln1- and cbln3-null mice have different phenotypes and cbln1 cbln3 double-null mice have deficits identical to those of cbln1 knockout mice. The basis for these discordant phenotypes is that Cbln1 and Cbln3 reciprocally regulate each other's degradation and secretion such that cbln1-null mice lack both Cbln1 and Cbln3, whereas cbln3-null mice lack Cbln3 but have an approximately sixfold increase in Cbln1. Unlike Cbln1, Cbln3 cannot form homomeric complexes and is secreted only when bound to Cbln1. Structural modeling and mutation analysis reveal that, by constituting a steric clash that is masked upon binding Cbln1 in a "hide-and-run" mechanism of endoplasmic reticulum retention, a single arginine confers the unique properties of Cbln3.     

Type 1 metabotropic glutamate receptors (mGlu1) trigger the gating of GluD2 delta glutamate receptors

The orphan GluD2 receptor belongs to the ionotropic glutamate receptor family but does not bind glutamate. Ligand-gated GluD2 currents have never been evidenced, and whether GluD2 operates as an ion channel has been a long-standing question. Here, we show that GluD2 gating is triggered by type 1 metabotropic glutamate receptors, both in a heterologous expression system and in Purkinje cells. Thus, GluD2 is not only an adhesion molecule at synapses but also works as a channel. This gating mechanism reveals new properties of glutamate receptors that emerge from their interaction and opens unexpected perspectives regarding synaptic transmission and plasticity.     

Primary structure and expression of the gamma 2 subunit of the glutamate receptor channel selective for kainate.

The presence and primary structure of a novel subunit of the mouse glutamate receptor channel, designated as gamma 2, have been revealed by cloning and sequencing the cDNA. The gamma 2 subunit has structural characteristics common to the neurotransmitter-gated ion channel family and shares a high amino acid sequence identity with the rat KA-1 subunit, thus constituting the gamma subfamily of the glutamate receptor channel. Expression of the gamma 2 subunit together with the beta 2 subunit in Xenopus oocytes yields functional glutamate receptor channels selective for kainate.