Ryoji Yano

Neuregulin-beta induces expression of an NMDA-receptor subunit.

Neuregulins (also known as ARIA, NDF, heregulin, GGF) are a family of widely expressed growth and differentiation factors. Neuregulins secreted from motor neurons accumulate at maturing neuromuscular junctions, where they stimulate transcription of genes encoding specific acetylcholine receptors. How these factors function at central synapses, however, is unknown. In the maturing cerebellum, neuregulins are concentrated in glutamatergic mossy fibres that innervate granule cells in the internal granule-cell layer. We have analysed the effects of neuregulins on the expression of genes encoding NMDA (N-methyl-D-aspartate) receptors in the cerebellum, because receptor composition changes dramatically as expression of the receptor NR2C subunit is specifically induced in neurons in the internal granule-cell layer during synaptogenesis. Here we report that addition of a neuregulin-β isoform to cultured cerebellar slices specifically increases the expression of NR2C messenger RNAs by at least 100-fold; effects are only minor with a neuregulin-α isoform. This stimulation of NR2C expression requires synaptic activity by NMDA receptors, as well as neuregulin-β. Addition of the NMDA-receptor-channel blocker AP-5 prevents upregulation of the NR2C subunit by neuregulin, whereas an AMPA/kainate-receptor antagonist does not. Consistent with these effects of neuregulin, we find that granule cells express its receptors ErbB2 and ErbB4 before the NR2C subunit of the NMDA receptor. Our results indicate that neuregulins regulate the composition of neurotransmitter receptors in maturing synapses in the brain, in a manner analogous to the neuromuscular junction.

Ethanol opens G-protein-activated inwardly rectifying K + channels

Ethanol affects many functions of the brain and peripheral organs. Here we show that ethanol opens G-protein-activated, inwardly rectifying K+ (GIRK) channels, which has important implications for inhibitory regulation of neuronal excitability and heart rate. At pharmacologically relevant concentrations, ethanol activated both brain-type GIRK1/2 and cardiac-type GIRK1/4 channels without interaction with G proteins or second messengers. Moreover, weaver mutant mice, which have a missense mutation in the GIRK2 channel, showed a loss of ethanol-induced analgesia. These results suggest that the GIRK channels in the brain and heart are important target sites for ethanol.

Functional coupling of the nociceptin/orphanin FQ receptor with the G-protein-activated K+ (GIRK) channel

Nociceptin/orphanin FQ is a heptadecapeptide which was recently isolated from brains. It induces hyperalgesia, in contrast to the analgesic effects of opioid ligands, although it and its receptor structurally resemble opioid peptides and opioid receptors, respectively. To investigate the molecular mechanism underlying nociceptin/orphanin FQ actions, we performed Xenopus oocyte expression assays, in situ hybridization histochemistry and electrophysiological analyses of neurons. We found that the nociceptin/orphanin FQ receptor is functionally coupled with the G-protein-activated K+ (GIRK) channel in Xenopus oocytes, and that the receptor mRNA and GIRK1 mRNA co-exist in various neurons, including hippocampal pyramidal cells. Furthermore, we found that nociceptin/orphanin FQ induces hyperpolarizing currents via inward-rectifier K+ channels in hippocampal pyramidal cells, suggesting that the nociceptin/orphanin FQ receptor couples with the GIRK channel in this region. We conclude that the nociceptin/orphanin FQ receptor couples with the GIRK channel in various neurons, including hippocampal pyramidal cells, thereby modulating neuronal excitability.

Distribution of prepro‐nociceptin/ orphanin FQ mRNA and its receptor mRNA in developing and adult mouse central nervous systems

Nociceptin/orphanin FQ (N/OFQ) and its receptor share similarities to opioids and their receptors in terms of the molecular structure and signaling pathway, but the two systems exhibit different actions in vivo. To understand the mechanism of N/OFQ‐system actions, we examined, by in situ hybridization analysis, the distribution of preproN/OFQ and N/OFQ receptor mRNAs in the developing and adult mouse central nervous systems (CNS). In most neural regions, preproN/OFQ mRNA was mainly expressed in a small population of middle‐sized neurons. These neurons were scattered between large projection‐type neurons or within the neuropil, suggestive of interneurons. In some other nuclei (lateral septum, bed nucleus of the stria terminalis, reticular thalamic nucleus, inferior colliculus, and rostral periolivery nucleus), preproN/OFQ mRNA was expressed in a number of large projection‐type neurons. By contrast, N/OFQ receptor mRNA was evenly expressed in most neurons of the adult CNS. Considering the inhibitory actions of N/OFQ, the distinct cellular expression pattern of the N/OFQ system suggests that the release of N/OFQ from interneurons may lower neuronal and synaptic activities of neighboring neurons, leading to integration or modulation of local circuits. Furthermore, the cellular expression pattern, distinct from that of the opioid system, may provide a possible molecular/cellular basis for the different in vivo actions of N/OFQ and opioids. In embryonic stages, both preproN/OFQ and N/OFQ receptor mRNAs were highly and widely expressed in the mantle zone, suggesting the possible importance of N/OFQ signaling in CNS development. J. Comp. Neurol. 399:139–151, 1998.

Molecular mechanisms of analgesia induced by opioids and ethanol: is the GIRK channel one of the keys?

Opioids and ethanol have been used since ancient times for pain relief. Opioid signaling is mediated by various effectors, including G protein-activated inwardly rectifying potassium (GIRK) channels, adenylyl cyclases, voltage-dependent calcium channels, phospholipase Cbeta(PLCbeta), and mitogen-activated protein kinases, although it has been unclear which effector mediates the analgesic effects of opioids. Ethanol induces a variety of physiological phenomena via various proteins, including GIRK channels rather than via membrane lipids. GIRK channel activation by either G proteins or ethanol is impaired in weaver mutant mice. The mutant mice may therefore serve as a useful animal model for studying the role of GIRK channels in vivo. Reduced analgesia by using either opioids or ethanol in weaver mutant mice suggests that GIRK channels are important effectors in both opioid- and ethanol-induced analgesia. This hypothesis is supported by similar findings in GIRK2 knockout mice. Among the various effectors coupled with opioid receptors and various targets of ethanol, GIRK channels are the only molecules whose involvement in opioid- and ethanol-induced analgesia has been demonstrated in vivo. The GIRK channel is potentially one of the key molecules in furthering the understanding of the pain control system and in developing advanced analgesics with fewer adverse effects.

Involvement of G-protein-activated inwardly rectifying K^+ (GIRK) channels in opioid-induced analgesia

To investigate the role of G-protein-activated inwardly rectifying K+ (GIRK) channels in opioid-induced analgesia, we compared the effects of opioids in wild-type and weaver mutant mice having mutant GIRK channels. In the tail-flick and hot-plate tests, weaver mutant mice displayed significantly lower analgesia after either morphine or (-)-U-50488 administration. These findings suggest that GIRK channel activation is important in the induction of analgesia by opioids.

Adaptor protein complex-4 (AP-4) is expressed in the central nervous system neurons and interacts with glutamate receptor δ2

Ion channels and receptors are targeted and localized at specific postsynaptic sites to mediate neurotransmission. Receptors clustering at postsynaptic sites has been extensively studied; however, the molecular mechanisms underlying intracellular trafficking of receptors to their specific destinations remain unclear. In the present study, we found that glutamate receptor delta2 interacted directly with AP-4, a newly identified adaptor protein complex-4 that mediates protein sorting in mammalian cells. The interaction between mu4 subunit of AP-4 and the delta2 C-terminal involved multiple amino acid sequence motifs other than the classical tyrosine-based signals. AP-4 complex is expressed ubiquitously in many regions of brain, with localization on the Golgi-like structures in the cell bodies and dendrites of neurons. In addition, overexpression of mu4 substantially altered the distribution pattern of delta2 in heterologous cells. These results suggest a potential involvement of AP-4 in the trafficking of delta2 in the brain.

Roles of neuregulin in synaptogenesis between mossy fibers and cerebellar granule cells

Neuregulins (NRGs), a large group of structurally related signaling proteins, are likely to have important roles in the development, maintenance and repair of the nervous system and other selected tissues. We have demonstrated, by using the major form of NRG cloned from the mouse cerebellum that both the soluble form and the membrane anchored form of NRG may serve different functions in synaptogenesis. The soluble form of NRG was produced by proteolytic cleavage of the membrane anchored form of NRG. The proteolytic cleavage was promoted by protein kinase activation. The cleaved form of NRG trans‐synaptically regulated the expression of the NMDA (N‐methyl‐D‐aspartate) receptor subunit NR2C as neurally‐derived factors, whereas the membrane anchored form of NRG showed a homophilic binding activity between NRGβ1s. In adult mice the membrane anchored form of NRG was concentrated in neuro‐terminals of both granule cells and pontocerebellar mossy fibers. The fact that NRG can be functionally viewed as cell recognition molecules as well as neurotrophic agents suggests new possibilities for the important class of molecules. J. Neurosci. Res. 59:612–623, 2000

Characterization of the apoptosis-associated tyrosine kinase (AATYK) expressed in the CNS

We isolated three related cDNA clones from a mouse cerebellar library; the type I cDNA was identical to the gene encoding the apoptosis-associated tyrosine kinase (AATYK), whose expression in myeloid precursor cells is increased during growth arrest or apoptosis. Low levels of AATYK mRNA expression were seen in adult mouse brains but not in embryos. In situ hybridization confirmed the widespread expression of AATYK mRNA in neurons throughout the adult brain. AATYK possessed tyrosine kinase activity and was autophosphorylated when expressed in 293 cells. AATYK mRNA expression was rapidly induced in cultured cerebellar granule cells during apoptosis induced by a low concentration of KCl (5 mM). Levels of endogenous AATYK protein were increased only slightly, but they were accompanied by an increase in molecular weight during apoptosis. Results of the tyrosine phosphatase treatments indicated that the increase in molecular weight was partly caused by tyrosine phosphorylation. The number of apoptotic granule cells overexpressing wild-type AATYK protein was significantly greater than the number of apoptotic granule cells overexpressing a mutant AATYK that lacked tyrosine kinase activity in low concentrations of KCl. These findings suggest that through its tyrosine kinase activity, AATYK is involved in the apoptosis of mature neurons.

PKC regulates the δ2 glutamate receptor interaction with S-SCAM/MAGI-2 protein

Abstract Inside cells, membrane proteins are localized at particular surface domains to perform their precise functions. Various kinds of PDZ domain proteins have been shown to play important roles in the intracellular trafficking and anchoring of membrane proteins. In this study, we show that δ2 glutamate receptor is interacting with S-SCAM/MAGI-2, a PDZ domain protein localized in the perinuclear region and postsynaptic sites of cerebellar Purkinje cells. The binding is regulated by PKC (protein kinase-C) mediated phosphorylation of the receptor with a unique repetitive structure in S-SCAM/MAGI-2. Co-expression of both proteins resulted in drastic changes of the receptor localization in COS7 cells. These results show a novel regulatory mechanism for the binding of PDZ domain proteins and suggest that the interaction between δ2 receptor and S-SCAM/MAGI-2 may be important for intracellular trafficking of the receptor.