Miwako Ozaki

Phenotypic Characterization of Transgenic Mice Overexpressing Neuregulin-1

Background Neuregulin-1 (NRG1) is one of the susceptibility genes for schizophrenia and implicated in the neurotrophic regulation of GABAergic and dopaminergic neurons, myelination, and NMDA receptor function. Postmortem studies often indicate a pathologic association of increased NRG1 expression or signaling with this illness. However, the psychobehavioral implication of NRG1 signaling has mainly been investigated using hypomorphic mutant mice for individual NRG1 splice variants. Methodology/Principal Findings To assess the behavioral impact of hyper NRG1 signaling, we generated and analyzed two independent mouse transgenic (Tg) lines carrying the transgene of green fluorescent protein (GFP)-tagged type-1 NRG1 cDNA. The promoter of elongation-factor 1α gene drove ubiquitous expression of GFP-tagged NRG1 in the whole brain. As compared to control littermates, both heterozygous NRG1-Tg lines showed increased locomotor activity, a nonsignificant trend toward decreasing prepulse inhibition, and decreased context-dependent fear learning but exhibited normal levels of tone-dependent learning. In addition, social interaction scores in both Tg lines were reduced in an isolation-induced resident-intruder test. There were also phenotypic increases in a GABAergic marker (parvalbumin) as well as in myelination markers (myelin basic protein and 2′,3′-cyclic nucleotide 3′-phosphodiesterase) in their frontal cortex, indicating the authenticity of NRG1 hyper-signaling, although there were marked decreases in tyrosine hydroxylase levels and dopamine content in the hippocampus. Conclusions These findings suggest that aberrant hyper-signals of NRG1 also disrupt various cognitive and behavioral processes. Thus, neuropathological implication of hyper NRG1 signaling in psychiatric diseases should be evaluated with further experimentation.

Transient exposure of neonatal mice to neuregulin-1 results in hyperdopaminergic states in adulthood: implication in neurodevelopmental hypothesis for schizophrenia

Neuregulin-1 (NRG1) is implicated in the etiology or pathology of schizophrenia, although its biological roles in this illness are not fully understood. Human midbrain dopaminergic neurons highly express NRG1 receptors (ErbB4). To test its neuropathological role in the neurodevelopmental hypothesis of schizophrenia, we administered type-1 NRG1 protein to neonatal mice and evaluated the immediate and subsequent effects on dopaminergic neurons and their associated behaviors. Peripheral NRG1 administration activated midbrain ErbB4 and elevated the expression, phosphorylation and enzyme activity of tyrosine hydroxylase (TH), which ultimately increased dopamine levels. The hyperdopaminergic state was sustained in the medial prefrontal cortex after puberty. There were marked increases in dopaminergic terminals and TH levels. In agreement, higher amounts of dopamine were released from this brain region of NRG1-treated mice following high potassium stimulation. Furthermore, NRG1-treated mice exhibited behavioral impairments in prepulse inhibition, latent inhibition, social behaviors and hypersensitivity to methamphetamine. However, there were no gross abnormalities in brain structures or other phenotypic features of neurons and glial cells. Collectively, our findings provide novel insights into neurotrophic contribution of NRG1 to dopaminergic maldevelopment and schizophrenia pathogenesis.

Protein processing and releases of neuregulin-1 are regulated in an activity-dependent manner.

Identification of the key molecules that bridge presynaptic neuronal events and long‐term modification of the postsynaptic process is an important challenge which will have to be met in order to further our understanding of the mechanisms for learning and memory. This study is focused on neuregulin‐1 (NRG‐1), a neurotrophic factor, that is known to regulate the development of various tissues and/or the life/death of cells through activation of the ErbB family receptor tyrosine kinases. It was discovered that the soluble form of NRG‐1 (sNRG‐1) is produced from the transmembrane form of NRG through proteolytic cleavage during electrical stimulation of either cultured cerebellar granule cells (GCs) or pontine nucleus neurons (PNs) that are presynaptic to GCs. sNRG‐1 was assayed by measuring the phosphorylation of both the ErbB receptors and cyclic AMP‐responsive element‐binding protein (CREB), and by means of antibodies to sNRG‐1. The cleavage and release of NRG‐1 depended on the frequency of electrical stimulation; the peak effect was at 50 Hz in both GCs and PNs. Activation of protein kinase C (PKC) mimicked this effect. The culture apparatus provided along with the mass‐electrical stimulation that was employed proved to be a powerful tool for combining neuronal electrical events and chemical events. We conclude from the results that, in mossy fibre (PN axon)‐GC synapses, electrical activity controls the proteolytic processing of NRG‐1 in a frequency‐dependent fashion and involves PKC. Furthermore, cleaved sNRG‐1 plays an important functional role in regulating transmission across these synapses.

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

Expression of receptors for neuregulins, ErbB2, ErbB3 and ErbB4, in developing mouse cerebellum

We have examined the expression of ErbB2, ErbB3 and ErbB4 in developing mouse cerebellum. ErbB2, ErbB3 and ErbB4 were all expressed in granule cells during cerebellar development. However, the expression pattern for each ErbB receptor changed with the developmental stage. Variations of signal transduction pathway through combinations of these ErbB receptors might have important roles in controlling cerebellar postnatal development.

G protein-activated inwardly rectifying K+ channel inhibition and rescue of weaver mouse motor functions by antidepressants.

Antidepressants, including tricyclic antidepressants (TCAs) and selective serotonin reuptake inhibitors (SSRIs), have been widely used for the treatment of not only depression but also other psychiatric disorders, although the molecular mechanisms of the drug effects have not yet been sufficiently revealed. Here, we investigated the in vivo effects of these antidepressants on G protein-activated inwardly rectifying K+ (GIRK) channels, which are important for regulating the excitability of various cells, by using weaver (wv) mice, which have mutant GIRK channels and show abnormal neuronal cell death and motor disturbances. First, we found that a widely used SSRI fluoxetine (also known as Prozac) effectively inhibited wv GIRK2 channels like wild-type GIRK channels, expressed in Xenopus oocytes. Next, we found that weaver motor disturbances were remarkably alleviated by chronic treatment with fluoxetine or desipramine. Furthermore, the chronic fluoxetine treatment substantially suppressed the abnormal neuronal cell death in the weaver mouse cerebellum and pontine nuclei. These results suggest that continuous inhibition of wv GIRK2 channels by a group of antidepressants caused substantial suppression of the neuronal cell death and resulted in improvement of motor abilities in weaver mice. These results provide evidence for in vivo GIRK channel inhibition by a group of antidepressants.

Degeneration of pontine mossy fibres during cerebellar development in weaver mutant mice

In weaver mutant mice, substitution of an amino acid residue in the pore region of GIRK2, a subtype of the G‐protein‐coupled inwardly rectifying K+ channel, changes the properties of the homomeric channel to produce a lethal depolarized state in cerebellar granule cells and dopaminergic neurons in substantia nigra. Degeneration of these types of neurons causes strong ataxia and Parkinsonian phenomena in the mutant mice, respectively. On the other hand, the mutant gene is also expressed in various other brain regions, in which the mutant may have effects on neuronal survival. Among these regions, we focused on the pontine nuclei, the origin of the pontocerebellar mossy fibres, projecting mainly into the central region of the cerebellar cortex. The results of histological analysis showed that by P9 the number of neurons in the nuclei was reduced in the mutant to about one half and by P18 to one third of those in the wild type, whereas until P7 the number were about the same in wild‐type and weaver mutant mice. Three‐dimensional reconstruction of the nuclei showed a marked reduction in volume and shape of the mutant nuclei, correlating well with the decrease in neuronal number. In addition, DiI (a lipophilic tracer dye) tracing experiments revealed retraction of pontocerebellar mossy fibres from the cerebellar cortex after P5. From these results, we conclude that projecting neurons in the pontine nuclei, as well as cerebellar granule cells and dopaminergic neurons in substantia nigra, strongly degenerate in weaver mutant mice, resulting in elimination of pontocerebellar mossy fibres during cerebellar development.

Analysis of patterned neuronal impulses and function of neuregulin.

Compared to other cells, except neural cells, the biggest property of neural cells is to have a particular electrical activity in each cell itself. The activity that shows a specific pattern will carry different information as a history of each neural cell. At present, we have examined the roles of neural impulses and revealed that a synaptic plasticity can be controlled by different patterned neural acitivites, such as different frequencies or oscillation patterns. Even though neural cells have similar genetic backgrounds, different environments give cells different neural activities and finally different characters of cells. Current studies have revealed that a particular pattern of neural activity, e.g. frequency, could be effective in some diseases. In response to environmental changes occurring throughout development and adult life, the brain reorganizes itself by adjusting the pattern of activity. In some cases, a particular pattern of neural acitivity decides the neural fate and should be able to control brain function even in higher functions. In the future, in order to understand the role of activity patterns and mechanisms of fundamental information processing in the brain, it will be necessary that the meaning of patterns is explained from molecular, biological and morphological perspectives, i.e., not only with metaphysical ‘phenomena’, but also at a physical ‘material’ level.

Imaging of molecular dynamics regulated by electrical activities in neural circuits and in synapses.

One of the major challenges in brain research is to unravel a network of molecules, neurons, circuits and systems that are responsible for dynamic and hierarchical brain functions. To understand molecular events that occur in synapses could be an important key to exploring the mechanism of information processing. A spatiotemporal recording method is required to observe neuronal activities in a particular local circuit and to resolve single synaptic potential with high resolution. As alternative methods, real-time imaging using fluorescent probes and optical recording methods are also a powerful approach for investigating the molecular dynamics of biological events in neurons in vitro and in vivo. Recently, optical imaging techniques have become of great importance to visualize the molecular dynamics in a micron-sized compartment of a single neuron such as neuronal synapse. In general, the presynaptic axon forms synapses at the postsynaptic site on the dendritic spines in the mammalian central nervous system. Subsets of the synapses undergo a series of enduring changes in spine shape and density as well as alterations in electrophysiological functions. Here we describe recent optical imaging studies conducted by elaborate methods and techniques that provide evidence for the link between neural activity and molecular dynamics.

Repeated administration of methamphetamine blocked cholecystokinin-octapeptide injection-induced c-fos mRNA expression without change in capsaicin-induced junD mRNA expression in rat cerebellum.

In the cerebellum, there are numerous cholecystokinin (CCK-8)-containing fibers. Since systemic CCK-8 injection-induced anxiety (psychological stress) activates the locus coeruleus cells that send mossy fiber inputs to the cerebellum, we examined whether systemic CCK-8 injections activate the rat and mouse cerebellum. First, injections of CCK-8 were found to induce c-fos mRNA expression in a vague patchy pattern that is different from single methamphetamine-induced Zebrin band-like c-fos mRNA expression, suggesting that the CCK-8 activating mossy fibers induce gene expression differently from the dopamine-containing mossy fibers in the ventral tegmental area. Second, since CCK-8 facilitates neural activity of dopamine in the midbrain, we examined whether repeated methamphetamine administration that induced behavioral sensitization had similar effects on the cerebellar CCK system. Repeated administration of methamphetamine suppressed the CCK-8-induced c-fos mRNA expression in the rat cerebellum. Third, capsaicin injections (physical stress) into a hind limb of the rat increased junD mRNA expression with no effect on c-fos mRNA expression, and repeated methamphetamine injections had no effect on the capsaicin-induced expression of junD mRNA. Fourth, either single injection of methamphetamine or CCK-8 to mice increased c-fos mRNA expression in the locus coeruleus, and so noradrenalin, but not dopamine, might interact with CCK-8-activating system. However, we considered the possibility unlikely. Thus, we conclude that repeated methamphetamine administration though dopamine selectively inhibits the c-fos mRNA expression after CCK-8 injection in the cerebellum.