Kazuaki Araki

Impairment of motor coordination, Purkinje cell synapse formation, and cerebellar long-term depression in GluRδ2 mutant mice

Of the six glutamate receptor (GluR) channel subunit families identified by molecular cloning, five have been shown to constitute either the AMPA, kainate, or NMDA receptor channel, whereas the function of the delta subunit family remains unknown. The selective localization of the delta 2 subunit of the GluR delta subfamily in cerebellar Purkinje cells prompted us to examine its possible physiological roles by the gene targeting technique. Analyses of the GluR delta 2 mutant mice reveal that the delta 2 subunit plays important roles in motor coordination, formation of parallel fiber-Purkinje cell synapses and climbing fiber-Purkinje cell synapses, and long-term depression of parallel fiber-Purkinje cell synaptic transmission. These results suggest a close relationship between synaptic plasticity and synapse formation in the cerebellum.

Cloning and expression of the ε4 subunit of the NMDA receptor channel

The primary structure of a novel subunit of the mouse NMDA (N‐methyl‐d‐aspartate) receptor channel, designated ε4, has been revealed by cloning and sequencing the cDNA. The ε4 subunit shares high amino acid sequence identity with the ε1, ε2 and ε3 subunits of the mouse NMDA. receptor channel, thus constituting the ε subfamily of the glutamate receptor channel. Expression from cloned cDNAs of the ε4 subunit together with the ζ1 subunit in Xenopus oocytes yields Functional NMDA receptor channels. The ε4/ζ1 heteromeric channel exhibits high apparent affinities for agonists and low sensitivities to competitive antagonists. The ε4 subunit is thus distinct in Functional properties from the ε1, ε2 and ε3 subunits, and contributes further diversity of the NMDA receptor channel.

Cloning, expression and modulation of a mouse NMDA receptor subunit

The primary structure and presence of two forms of the mouse N‐methyl‐d‐aspartate (NMDA) receptor channel subunit ζl have been disclosed by cloning and sequencing the cDNAs. The ζl subunit shows −20% amino acid sequence identities with the rodent α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA)‐ or kainate‐selective GluR subunits and has structural features common to neurotransmitter‐gated ion channels. Functional homomeric ζl channels expressed in Xenopus oocytes by injection of the subunit specific mRNA exhibit current responses characteristics for the NMDA receptor channel such as activation by glycine, Ca2+ permeability, blocking by Mg2+ and activation by polyamine. It has been found that the ζl channel activity is positively modulated by treatment with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA).

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.

Molecular cloning of a cDNA encoding a novel member of the mouse glutamate receptor channel family

The primary structure of a novel putative subunit of the mouse glutamate receptor channel, designated as delta 1, has been deduced by cloning and sequencing the cDNA. The delta 1 subunit shows 21-25% amino acid sequence identity with previously characterized rodent glutamate receptor channel subunits and thus may represent a new subfamily of the glutamate receptor channel.

Reduced spontaneous activity of mice defective in the ε4 subunit of the NMDA receptor channel

In an attempt to examine the functional significance of the molecular diversity of theN-methyl-d-aspartate (NMDA) receptor channel, we generated mutant mice defective in the e4 subunit by gene targeting technique. The e4 subunit mutant mice grew and mated normally. No e4 subunit protein was detected in the homozygous mutant mice, and the amount of the e4 subunit protein of 155 kDa was reduced in the heterozygous mice. The expressions of the other NMDA receptor channel subunit mRNAs were not appreciably affected by the mutation. The mutant mice exhibited no obvious histological abnormalities in the various brain regions and in the formation of whisker-related neuronal patterns (barrels, barreloids and barrelettes). In an open field test, however, the e4 subunit mutant mice showed a reduced spontaneous activity. No significant difference was found between the heterozygous and mutant mice in motor activity and anxiety tests. These results suggest that the e4 subunit of the NMDA receptor channel plays a role in vivo in controlling the spontaneous behavioral activity.

Brain-derived neurotrophic factor regulates the expression of AMPA receptor proteins in neocortical neurons.

The role of the neurotrophins; nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5, in synaptic development and plasticity has been extensively investigated. The neurotrophins regulate synaptic transmission as well as neural development in the brain. However, the mechanisms underlying these processes are unknown. In this study we show that brain-derived neurotrophic factor triggers an increase in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor (GluR) proteins without significant changes in their messenger RNA levels. Brain-derived neurotrophic factor treatment specifically increased the protein levels of GluR1 (193+/-22%) and GluR2/3 (182+/-11%) in cultured rat neocortical neurons. In contrast, nerve growth factor and neurotrophin-3 failed to alter the protein levels of these neurons, and brain-derived neurotrophic factor effects on N-methyl-D-aspartate-type glutamate receptors were either modest or negligible. Immunocytochemical studies indicated that the increase in AMPA receptor proteins reflects the induction of their neuronal expression, but not selective neuronal survival. In agreement with these results, cortical neurons from brain-derived neurotrophic factor-knockout mice exhibited a reduction in AMPA receptor proteins in the cytoskeletal fraction containing postsynaptic proteins. Thus, the neurotrophin plays a crucial role in modulating the expression of AMPA receptors presumably at translational or post-translation levels and is implicated in synaptic development and plasticity.

Prefrontal Abnormality of Schizophrenia Revealed by DNA Microarray: Impact on Glial and Neurotrophic Gene Expression

Abstract: DNA microarrays with isotope labeling from gene‐specific primers enable sensitive detection of rare mRNAs, including neurotrophin and cytokine mRNAs in the brain. Using high‐quality RNA from postmortem brains, gene‐expression profiles covering 1373 genes were assessed in the dorsoprefrontal cortex of schizophrenic patients and compared with those of nonpsychiatric subjects. Statistical analysis of the DNA microarray data confirmed the findings of a previous GeneChip study by Hakak et al. (Proc. Natl. Acad. Sci. USA Vol. 98, pp. 4746‐4751, 2001). The highest frequency of mRNA expression alterations occurred in oligodendrocyte‐ and astrocyte‐related genes in the prefrontal cortex of schizophrenic patients, followed by the category for the genes for growth factors/neurotrophic factors and their receptors. Whether each mRNA signal represents the expression of the individual genes or homologous genes in the category remains to be determined, however. To control for potential medication effects on patients, RNA from cynomolgus monkeys that were treated with haloperidol for 3 months was also subjected to DNA microarray analysis. A few genes overlapped between the gene‐expression profiles of the monkeys and patients. The present profiling study suggests a potential biological link between abnormal neurotrophic signals and impaired glial functions in schizophrenic pathology.

Suppression of LTD in cultured Purkinje cells deficient in the glutamate receptor δ2 subunit

Involvement of the glutamate receptor channel delta 2 subunit in cerebellar long-term depression (LTD) was studied in cultures prepared from wild-type and mutant mice deficient in the delta 2 subunit. LTD of the glutamate response was induced by pairing glutamate applications and depolarization of a Purkinje cell in wild-type culture. However, in cultured Purkinje cells prepared from mutant mice, the same conditioning failed to induce LTD. Immunocytological staining showed that mutant Purkinje cells develop and express calbindin (a marker protein for Purkinje cells) as do wild-type cells, but they express no delta 2 subunit protein. The results indicate that the glutamate receptor channel delta 2 subunit is involved in the postsynaptic down-regulation of glutamate sensitivity, presumably during cerebellar LTD.

Involvement of the glutamate receptor δ2 subunit in the long-term depression of glutamate responsiveness in cultured rat Purkinje cells

An antisense oligonucleotide against the glutamate receptor delta 2 subunit mRNA, which is selectively expressed only in Purkinje neurons, suppressed the induction of long-term depression (LTD) of glutamate responsiveness in the rat cerebellar culture. LTD of glutamate response is induced by pairing glutamate application and depolarization of a Purkinje cell. Treatment of the culture with the antisense oligonucleotide exerted no appreciable effect on basic physiological and morphological properties of Purkinje cells, except for LTD induction and reduction of delta immunoreactivity which was intense in distal dendrites. Sense and missense oligonucleotides, which were used as controls, did not block LTD induction. These results suggest that the glutamate receptor delta 2 subunit is involved in the cerebellar LTD.