Etsuko Kushiya

Impairment of suckling response, trigeminal neuronal pattern formation, and hippocampal LTD in NMDA receptor ε2 subunit mutant mice

Multiple epsilon subunits are major determinants of the NMDA receptor channel diversity. Based on their functional properties in vitro and distributions, we have proposed that the epsilon 1 and epsilon 2 subunits play a role in synaptic plasticity. To investigate the physiological significance of the NMDA receptor channel diversity, we generated mutant mice defective in the epsilon 2 subunit. These mice showed no suckling response and died shortly after birth but could survive by hand feeding. The mutation hindered the formation of the whisker-related neuronal barrelette structure and the clustering of primary sensory afferent terminals in the brainstem trigeminal nucleus. In the hippocampus of the mutant mice, synaptic NMDA responses and longterm depression were abolished. These results suggest that the epsilon 2 subunit plays an essential role in both neuronal pattern formation and synaptic plasticity.

Functional expression from cloned cDNAs of glutamate receptor species responsive to kainate and quisqualate

The complete amino acid sequences of two mouse glutamate receptor subunits (GluR1 and GluR2) have been deduced by cloning and sequencing the cDNAs. Xenopus oocytes injected with mRNA derived from the GluR1 cDNA exhibit current responses both to kainate and to quisqualate as well as to glutamate, whereas oocytes injected with mRNA derived from the GluR2 cDNA show little response. Injection of oocytes with both the mRNAs produces current responses larger than those induced by the GluR1‐specific mRNA and the dose‐response relations indicate a positively cooperative interaction between the two subunits. These results suggest that kainate and quisqualate can activate a common glutamate receptor subtype and that glutamate‐gated ionic channels are hetero‐oligomers of different subunits.

Primary structure and expression of the γ2 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.

Role of the Carboxy-Terminal Region of the GluRε2 Subunit in Synaptic Localization of the NMDA Receptor Channel

The synaptic localization of the N-methyl-D-aspartate (NMDA) type glutamate receptor (GluR) channel is a prerequisite for synaptic plasticity in the brain. We generated mutant mice carrying the carboxy-terminal truncated GluR epsilon2 subunit of the NMDA receptor channel. The mutant mice died neonatally and failed to form barrelette structures in the brainstem. The mutation greatly decreased the NMDA receptor-mediated component of hippocampal excitatory postsynaptic potentials and punctate immunofluorescent labelings of GluR epsilon2 protein in the neuropil regions, while GluR epsilon2 protein expression was comparable. Immunostaining of cultured cerebral neurons showed the reduced punctate staining of the truncated GluR epsilon2 protein at synapses. These results suggest that the carboxy-terminal region of the GluRepsilon2 subunit is important for efficient clustering and synaptic localization of the NMDA receptor channel.

Gq protein alpha subunits Galphaq and Galpha11 are localized at postsynaptic extra-junctional membrane of cerebellar Purkinje cells and hippocampal pyramidal cells.

Following cell surface receptor activation, the α subunit of the Gq subclass of GTP‐binding proteins activates the phosphoinositide signalling pathway. Here we examined the expression and localization of Gq protein α subunits in the adult mouse brain by in situ hybridization and immunohistochemistry. Of the four members of the Gq protein α subunits, Gαq and Gα11 were transcribed predominantly in the brain. The highest transcriptional level of Gαq was observed in cerebellar Purkinje cells (PCs) and hippocampal pyramidal cells, while that of Gα11 was noted in hippocampal pyramidal cells. Antibody against the C‐terminal peptide common to Gαq and Gα11 strongly labelled the cerebellar molecular layer and hippocampal neuropil layers. In these regions, immunogold preferentially labelled the cytoplasmic face of postsynaptic cell membrane of PCs and pyramidal cells. Immunoparticles were distributed along the extra‐junctional cell membrane of spines, dendrites and somata, but were almost excluded from the junctional membrane. By double immunofluorescence, Gαq/Gα11 was extensively colocalized with metabotropic glutamate receptor mGluR1α in dendritic spines of PCs and with mGluR5 in those of hippocampal pyramidal cells. Together with concentrated localization of mGluR1α and mGluR5 in a peri‐junctional annulus on PC and pyramidal cell synapses ( Baude et al. 1993 , Neuron, 11, 771–787; Luján et al. 1996 , Eur. J. Neurosci., 8, 1488–1500), the present molecular‐anatomical findings suggest that peri‐junctional stimulation of the group I metabotropic glutamate receptors is mediated by Gαq and/or Gα11, leading to the activation of the intracellular effector, phospholipase Cβ.

Abundant distribution of TARP γ-8 in synaptic and extrasynaptic surface of hippocampal neurons and its major role in AMPA receptor expression on spines and dendrites

Transmembrane α‐amino‐3‐hydroxyl‐5‐isoxazolepropionate (AMPA) receptor regulatory proteins (TARPs) play pivotal roles in AMPA receptor trafficking and gating. Here we examined cellular and subcellular distribution of TARP γ‐8 in the mouse brain. Immunoblot and immunofluorescence revealed the highest concentration of γ‐8 in the hippocampus. Immunogold electron microscopy demonstrated dense distribution of γ‐8 on the synaptic and extrasynaptic surface of hippocampal neurons with very low intracellular labeling. Of the neuronal surface, γ‐8 was distributed at the highest level on asymmetrical synapses of pyramidal cells and interneurons, whereas their symmetrical synapses selectively lacked immunogold labeling. Then, the role of γ‐8 in AMPA receptor expression was pursued in the hippocampus using mutant mice defective in the γ‐8 gene. In the mutant cornu ammonis (CA)1 region, synaptic and extrasynaptic AMPA receptors on dendrites and spines were severely reduced to 35–37% of control levels, whereas reduction was mild for extrasynaptic receptors on somata (74%) and no significant decrease was seen for intracellular receptors within spines. In the mutant CA3 region, synaptic AMPA receptors were reduced mildly at asymmetrical synapses in the stratum radiatum (67% of control level), and showed no significant decrease at mossy fiber–CA3 synapses. Therefore, γ‐8 is abundantly distributed on hippocampal excitatory synapses and extrasynaptic membranes, and plays an important role in increasing the number of synaptic and extrasynaptic AMPA receptors on dendrites and spines, particularly, in the CA1 region. Variable degrees of reduction further suggest that other TARPs may also mediate this function at different potencies depending on hippocampal subregions, input sources and neuronal compartments.

A single amino acid residue determines the Ca2+ permeability of AMPA-selective glutamate receptor channels

Functional analysis of AMPA-selective glutamate receptor channels expressed in Xenopus oocytes from cloned cDNAs has shown that homomeric channels formed by the GluR1 subunit are permeable to Ca2+, whereas heteromeric channels composed of the GluR1 and GluR2 subunits show little permeability. Furthermore, substitution of glutamine for arginine in putative transmembrane segment M2 of the GluR2 subunit makes the heteromeric channels permeable to Ca2+. These results suggest that the GluR2 subunit plays a key role in keeping AMPA-selective glutamate receptor channels essentially impermeable to Ca2+ and that the critical determinant is the positively charged residue in M2 segment.

cDNA cloning and nucleotide sequence of rat muscle-specific enolase (ββ enolase)

The nucleotide sequence of rat muscle‐specific enolase cDNA was determined by sequencing three cDNA clones encoding this enolase isozyme. The nearly full‐length cDNA consists of 13‐bp 5′‐ and 84‐bp 3′‐noncoding regions and a poly(A) tail in addition to a 1302‐bp coding region encoding a polypeptide composed of 434 amino acid residues. The deduced primary structure of this enolase isozyme is about 80% similar to those determined previously for rat neuron‐specific and non‐neuronal enolase isozymes. Southern blot analysis suggested strongly the existence of a single copy of the muscle‐specific enolase gene per haploid genome. The mRNA for this enolase isozyme was detected in rat skeletal muscle on day 1 after birth and its level increased rapidly during 10–30 days after birth without any change in its size (1500 bases).

Cloning and functional expression of a cDNA encoding the mouse β2 subunit of the kainate-selective glutamate receptor channel

The primary structure of the mouse glutamate receptor beta 2 subunit has been deduced by cloning and sequencing cDNA. The beta 2 subunit has structural characteristics common to the subunits of glutamate-gated ion channels. Expression of the cloned cDNA in Xenopus oocytes yields functional glutamate receptor channels selective for kainate.

Partial Purification and Characterization of Messenger RNA Coding 14-3-2 Protein from Rat Brain

Abstract: 14‐3‐2 Protein (neuron‐specific enolase) is a neuron‐specific protein. Using a reticulocyte lysate cell‐free system for translation of 14‐3‐2 protein mRNA, we have partially purified this mRNA by several procedures, including formamide sucrose density centrifugation, formamide polyacrylamide gel electrophoresis (PAGE) and polyuridylic acid (poly(U))‐Sepharose affinity chromatography. Using mRNA obtained by these procedures, we could increase the translation ratio of 14‐3‐2 protein synthesized/total soluble protein synthesized to 7.31%. The overall purification was 37.8‐fold. The size of 14‐3‐2 protein mRNA appears to be about 19–20S, because translation activity of mRNA obtained by sucrose density gradient centrifugation or formamide PAGE was the most active in this RNA size.