Glutamate Receptor Delta2 Subunit Research Articles

Glutamate receptors of the delta family are widely expressed in the adult brain.

Recent reports point to critical roles of glutamate receptor subunit delta2 (GluD2) at excitatory synapses and link GluD1 gene alteration to schizophrenia but the expression patterns of these subunits in the brain remain almost uncharacterized. We examined the distribution of GluD1-2 mRNAs and proteins in the adult rodent brain, focusing mainly on GluD1. In situ hybridization revealed widespread neuronal expression of the GluD1 mRNA, with higher levels occurring in several forebrain regions and lower levels in cerebellum. Quantitative RT-PCR assessed differential GluD1 expression in cortex and cerebellum, and revealed GluD2 expression in cortex, albeit at markedly lower level than in cerebellum. Likewise, a high GluD1/GluD2 mRNA ratio was observed in cortex and a low ratio in cerebellum. GluD1 and GluD2 mRNAs were co-expressed in single cortical and hippocampal neurons, with a large predominance of GluD1. Western blots using GluD1- and GluD2-specific antibodies showed expression of both subunits in various brain structures, but not in non-nervous tissues examined. Both delta subunits were upregulated during postnatal development. Widespread neuronal expression of the GluD1 protein was confirmed using immunohistochemistry. Examination at the electron microscopic level in the hippocampus revealed that GluD1 was mainly localized at postsynaptic density of excitatory synapses on pyramidal cells. Control experiments performed using mice carrying deletion of the GluD1- or the GluD2-encoding gene confirmed the specificity of the present mRNA and protein analyses. Our results support a role for the delta family of glutamate receptors at excitatory synapses in neuronal networks throughout the adult brain.

De novo partial deletion in GRID2 presenting with complicated spastic paraplegia

Complex forms of spastic paraplegia (SPG) are rare and genetically heterogeneous. In apparently sporadic cases, analysis of known SPG genes often fails to reveal a mutation. We report a 24-year-old patient with a syndrome of spastic paraplegia, ataxia, frontotemporal dementia, and lower motor neuron involvement. Screening of the patient's genome for copy number variation identified a novel 276 kb deletion spanning the first exon of the GRID2 gene. MRI scan showed atrophy of the cerebellum, and electromyography revealed a chronic disorder of motor neurons or their axons. A deletion in GRID2, coding for the glutamate receptor delta-2 subunit precursor protein, was excluded in either parent, suggesting that the deletion in the index patient occurred de novo. We hypothesize that the deletion identified here is the cause of our patient's clinical presentation, due to the resemblance to the GRID2 mutation phenotype in mouse models.

A Homozygous Deletion in GRID2 Causes a Human Phenotype With Cerebellar Ataxia and Atrophy

GRID2 is a member of the ionotropic glutamate receptor family of excitatory neurotransmitter receptors. GRID2 encodes the glutamate receptor subunit delta-2, selectively expressed in cerebellar Purkinje cells. The phenotype associated with loss of GRID2 function was described only in mice until now, characterized by different degrees of cerebellar ataxia and usually relatively mild abnormalities of the cerebellum. This work describes for the first time the human phenotype associated with homozygous partial deletion of GRID2 in 3 children in one large consanguineous Turkish family. Homozygous deletion of exons 3 and 4 of GRID2 (94153589-94298037bp) in the proband and similarly affected cousins, and heterozygous deletions in parental DNA were shown using Affymetrix® 6.0 single-nucleotide polymorphism array, confirmed by real-time polymerase chain reaction. The phenotype includes nystagmus, hypotonia with marked developmental delay in gross motor skills in early infancy followed by a static encephalopathy course with development of cerebellar ataxia, oculomotor apraxia, and pyramidal tract involvement.

The glutamate receptor subunit delta2 is capable of gating its intrinsic ion channel as revealed by ligand binding domain transplantation

The family of ionotropic glutamate receptors includes 2 subunits, delta1 and delta2, the physiological relevance of which remains poorly understood. Both are nonfunctional in heterologous expression systems, although the isolated, crystallized ligand binding domain (LBD) of delta2 is capable of binding D-serine. To investigate these seemingly contradictory observations we tested whether delta receptors can be ligand gated at all. We used a strategy that replaced the native LBD of delta2 by a proven glutamate-binding LBD. Test transplantations between alpha-amino-3-hydroxy-5-methylisoxazole propionate (AMPA) and kainate receptors (GluR1 and GluR6, respectively) showed that this approach can produce functional chimeras even if only one part of the bipartite LBD is swapped. Upon outfitting delta2 with the LBD of GluR6, the chimera formed glutamate-gated ion channels with low Ca(2+) permeability and unique rectification properties. Ligand-induced conformational changes can thus gate delta2, suggesting that the LBD of this receptor works fundamentally differently from that of other ionotropic glutamate receptors.

What Does it Take to Gate AMPA Receptors?

In vivo, agonist binding to the open conformation of the ligand binding domain initiates the process of gating in ionotropic glutamate receptors. Arguably, an alternative manner to gate the receptors exists, which requires a point mutation in the most-conserved sequence motif in the second transmembrane domain. Originally, this mutation occurred spontaneously in the orphan glutamate receptor subunit delta2, causing the ataxic phenotype of lurcher mice.1 In the absence of a ligand that could initiate gating at this orphan subunit, the introduction of the lurcher mutation led to spontaneous currents through delta2-lurcher channels.1 Introduction of the corresponding mutation into the AMPA receptor GluR1 induced a number of aberrant gating properties.2-5 Among those, glutamate potency was highly increased, and competitive antagonists suddenly behaved as partial agonists.2,5 We reported that the introduction of delta2 amino acids in the domain preceding the first transmembrane domain in GluR1 resulted in a mutant receptor that displayed all characteristics of lurcher-typical gating. We proposed that lurcher-like mutations work to enhance gating by destabilizing the closed state of the receptor. As a result, no or minimal conformational changes in the ligand binding domain are sufficient for gating, explaining, respectively, why spontaneous currents and competitive antagonists act as partial agonists in lurcher-like channels. Strikingly, a similar conversion of antagonists upon coexpression of TARPs with glutamate receptors has recently been reported.6,7 We take this as indication that the actual mechanism of action might be very similar, and that both lurcher-like mutations and TARPs work as 'gating enhancers'.

Defective control and adaptation of reflex eye movements in mutant mice deficient in either the glutamate receptor δ2 subunit or Purkinje cells

The ionotropic glutamate receptor delta2 subunit (GluRdelta2) is selectively expressed in cerebellar Purkinje cells and is implicated in long-term depression, synaptic formation and elimination. To study the effect of GluRdelta2 deficiency on motor control, we measured the vestibulo-ocular reflex (VOR) and optokinetic response (OKR) induced by sinusoidal rotation of the animal and/or the surrounding screen in two GluRdelta2 mutant mice: a GluRdelta2 knockout mouse (delta2-/-) and a lurcher mouse with a point mutation in the GluRdelta2 gene resulting in loss of all Purkinje cells. delta2-/- showed significantly higher VOR gain in the dark (VORD) than in the wild-type. In delta2-/-, the VOR gain in light was lower than that in the dark. The phase of OKR lagged more in delta2-/- than in lurcher and wild-type mice. Both mutant mice failed to change the VORD or OKR gain adaptively in response to sustained vestibular and/or visual stimulation. Basal properties of VOR and OKR changed little by lesion of the flocculus, but they changed substantially by lesion of the inferior olivary nuclei (IO). The abnormal VOR gain and OKR phase delay were clearly reduced in delta2-/- by the latter lesion. Our results indicate that failures in the GluRdelta2-dependent synaptic regulation affect motor performance more severely than loss of cerebellar cortical outputs. This study suggests that the anomalies in delta2-/- are dependent on inputs from IO and that GluRdelta2 deficiency changed properties of not only the cerebellar cortex but also the brainstem neuronal pathways controlling reflex eye movements during development.

Oscillating Purkinje neuron activity causing involuntary eye movement in a mutant mouse deficient in the glutamate receptor delta2 subunit.

How failures in regulation of synaptic transmission in the mammalian CNS affect neuronal activity and disturb motor coordination is addressed. The mutant mouse deficient in the glutamate receptor delta2 subunit, specifically expressed in cerebellar Purkinje neurons, has defects in synaptic regulations such as synaptic plasticity, stabilization, and elimination of synaptic connections and shows failures in motor coordination and learning. In this study, the cause of motor discoordination of the delta2 mutant mouse was analyzed by comparing its motor control ability with those of the wild-type mouse and the lurcher mutant mouse, which loses all Purkinje neurons, the sole output neurons in the cerebellar cortex. Unexpectedly, the delta2 mutant mouse showed severer motor discoordination than the lurcher mouse without any cerebellar cortical outputs. The delta2 mutant mouse showed involuntary spontaneous eye movement with characteristic 10 Hz oscillation, which disappeared by ablation of the cerebellar flocculus, suggesting that the delta2 mutant cerebellar cortex outputs an abnormal signal. In vivo extracellular recordings of neuronal activity revealed that Purkinje neurons tended to fire clustered action potentials and complex spikes at approximately 10 Hz in the delta2 mutant mouse. A whole-cell patch-clamp recording from Purkinje neurons in cerebellar slices indicated that the clustered action potentials could be induced by climbing fiber activation. Taken together, our results suggest that the delta2 subunit deficiency produces the oscillating activity in Purkinje neurons by enhancing climbing fiber inputs, causing surplus movement and affecting motor control worse than no signal at all.

Identification and characterization of human GRID2IP gene and rat Grid2ip gene in silico

Formin-homology proteins are implicated in the cell polarity control through the assembly of specific actin structures. FMNL1/KW-13/FMNL, FMNL2/KIAA1902/FHOD2, FMNL3/KIAA2014, DAAM1, DAAM2, DIAPH1 and DIAPH2 are Formin-homology proteins with the FDD domain, while Fmn1, Fmn2, FHOD1 and Grid2ip/Delphilin are Formin-homology proteins without the FDD domain. Mouse Grid2ip links glutamate receptor delta2 subunit with actin cytoskeleton and various signaling molecules. Here, we identified and characterized human GRID2IP gene as well as rat Grid2ip gene by using bioinformatics. Human GRID2IP gene was identified within human genome sequence CTD-2195F21 (AC072052.6). Human GRID2IP gene, consisting of 21 exons, was mapped to human chromosome 7p22.1. Rat Grid2ip gene, consisting of 21 exons, was identified within rat genome sequence CH230-82F18 (AC126572.3). Human GRID2IP (1020 aa) showed 91.7% total-amino-acid identity with rat Grid2ip (1024 aa), and 92.7% total-amino-acid identity with mouse Grid2ip. Human GRID2IP protein was found to consist of PDZ domain (codon 94-166), GRCAH domain (codon 204-269), FH1 domain (codon 559-621), and FH2 domain (codon 640-1005). GRCAH domain identified in this study was conserved among mammalian GRID2IP orthologs and mammalian CIP98/KIAA1526 orthologs. This is the first report on comprehensive characterization of human GRID2IP gene as well as on identification of GRCAH domain.

The Hippocampus Plays an Important Role in Eyeblink Conditioning with a Short Trace Interval in Glutamate Receptor Subunit δ2 Mutant Mice

Mutant mice lacking the glutamate receptor subunit delta2 exhibit changes in the structure and function of the cerebellar cortex. The most prominent functional feature is a deficiency in the long-term depression (LTD) at parallel fiber-Purkinje cell synapses. These mutant mice exhibit severe impairment during delay eyeblink conditioning but learn normally during trace eyeblink conditioning without the cerebellar LTD, even with a 0 trace interval. We investigated the hippocampal contribution to this cerebellar LTD-independent "0 trace interval" learning. The mutant mice whose dorsal hippocampi were aspirated exhibited severe impairment in learning, whereas those that received post-training hippocampal lesions retained the memory. The wild-type mice showed no impairment in either case. These results suggest that the hippocampal component of the eyeblink conditioning task becomes dominant when cerebellar LTD is impaired.

Impairment of eyeblink conditioning in GluRdelta2-mutant mice depends on the temporal overlap between conditioned and unconditioned stimuli.

Mice lacking the glutamate receptor subunit delta2 (GluRdelta2) are deficient in cerebellar long-term depression (LTD) at the parallel fibre-Purkinje cell synapses. We conducted delay and trace eyeblink conditioning with these mice, using various temporal intervals between the conditioned stimulus (CS) and unconditioned stimulus (US). During trace conditioning in which a stimulus-free trace interval (TI) of 250, 100 or 50 ms intervened between the 352-ms tone CS and 100-ms US, GluRdelta2-mutant mice learned as successfully as wild-type mice. Even in the paradigm with TI = 0 ms, in which the end of CS and onset of US are simultaneous, there was no difference between the GluRdelta2-mutant and wild-type mice in their acquisition of a conditioned response. However, in the delay paradigm in which the 452-ms CS overlapped temporally with the coterminating 100-ms US, GluRdelta2-mutant mice exhibited severe learning impairment. The present study together with our previous work [Kishimoto, Y., Kawahara, S., Suzuki, M., Mori, H., Mishina, M. & Kirino, Y. (2001) Eur. J. Neurosci., 13, 1249-1254], indicates that cerebellar LTD-independent learning is possible in paradigms without temporal overlap between the CS and US. On the other hand, GluRdelta2 and cerebellar LTD are essential for learning when there is CS-US temporal overlap, suggesting that the cerebellar neural substrates underlying eyeblink conditioning may change, depending on the temporal overlap of the CS and US.

Identification of vestibular compensation-associated molecules by means of differential display

The differential display method was used to identify gene expression which is altered in the cerebellar flocculus after unilateral labyrinthectomy (UL). Total RNA from flocculi of sham-operated and labyrinthectomized rats was isolated, amplified by PCR using arbitrary primer sets and separated by electrophoresis on a polyacrylamide gel. PCR products, whose amounts were significantly different in samples from labyrinthectomized animals and those from controls, were cut out of the gel and sequenced. One of the up-regulated products was the rat protein phosphatase 2A beta catalytic subunit mRNA and one of the down-regulated products was the rat glutamate receptor delta-2 subunit mRNA. Histochemical examination of in situ hybridization showed that those molecules were intensively localized in the Purkinje cell layer. In labyrinthectomized rats, UL-induced nystagmus gradually disappeared within 3 days after UL. These findings suggest that changes in expression of those molecules in the floccular Purkinje cells after UL is involved in vestibular compensation. So far various kinds of neural plasticity-associated molecules have been investigated, mainly by slice-in vitro studies. This study indicates that differential display is a feasible molecular biological in vivo method for investigation of the mechanism of neural plasticity.