Yuji Kiyama

Interactions between plexin-A2, plexin-A4, and semaphorin 6A control lamina-restricted projection of hippocampal mossy fibers.

Hippocampal mossy fibers project preferentially to the stratum lucidum, the proximal-most lamina of the suprapyramidal region of CA3. The molecular mechanisms that govern this lamina-restricted projection are still unknown. We examined the projection pattern of mossy fibers in mutant mice for semaphorin receptors plexin-A2 and plexin-A4, and their ligand, the transmembrane semaphorin Sema6A. We found that plexin-A2 deficiency causes a shift of mossy fibers from the suprapyramidal region to the infra- and intrapyramidal regions, while plexin-A4 deficiency induces inappropriate spreading of mossy fibers within CA3. We also report that the plexin-A2 loss-of-function phenotype is genetically suppressed by Sema6A loss of function. Based on these results, we propose a model for the lamina-restricted projection of mossy fibers: the expression of plexin-A4 on mossy fibers prevents them from entering the Sema6A-expressing suprapyramidal region of CA3 and restricts them to the proximal-most part, where Sema6A repulsive activity is attenuated by plexin-A2.

Differential expression of two zebrafish emx homeoprotein mRNAs in the developing brain

The complete amino-acid sequences of two zebrafish emx homeoproteins containing a homeodomain homologous to that of the Drosophila empty spiracles head gap gene product have been determined by cloning and sequencing of cDNAs. The zebrafish emx1 and emx2 homeoproteins consist of 233 and 247 amino acids, respectively. The zebrafish emx1 and emx2 mRNAs are present at 12 h after fertilization, when the presumptive brain is in a simple tubular structure, and before first primary neurons appear. During brain development, the emx1 mRNA is localized in the dorsal telencephalon, whereas the emx2 mRNA is distributed in the dorsal telencephalon, parts of the diencephalon and the otocyst. The differential expression patterns of the two emx homeoprotein mRNAs may define the subdivisions of the zebrafish telencephalon.

NR2B tyrosine phosphorylation modulates fear learning as well as amygdaloid synaptic plasticity

Phosphorylation of neural proteins in response to a diverse array of external stimuli is one of the main mechanisms underlying dynamic changes in neural circuitry. The NR2B subunit of the NMDA receptor is tyrosine‐phosphorylated in the brain, with Tyr‐1472 its major phosphorylation site. Here, we generate mice with a knockin mutation of the Tyr‐1472 site to phenylalanine (Y1472F) and show that Tyr‐1472 phosphorylation is essential for fear learning and amygdaloid synaptic plasticity. The knockin mice show impaired fear‐related learning and reduced amygdaloid long‐term potentiation. NMDA receptor‐mediated CaMKII signaling is impaired in YF/YF mice. Electron microscopic analyses reveal that the Y1472F mutant of the NR2B subunit shows improper localization at synapses in the amygdala. We thus identify Tyr‐1472 phosphorylation as a key mediator of fear learning and amygdaloid synaptic plasticity.

Enhancement of hippocampal LTP, reference memory and sensorimotor gating in mutant mice lacking a telencephalon-specific cell adhesion molecule.

Telencephalin (TLCN) is a cell adhesion molecule selectively expressed in the telencephalon of the mammalian brain. The mutant mice lacking TLCN had no detectable abnormalities in their neural development and synaptic structures. Ablation of TLCN increased the hippocampal long‐term potentiation and its saturation level. The TLCN mutation selectively enhanced the performance of the radial maze and water‐finding tasks, learning tasks with appetitive reinforcers, but not the contextual fear conditioning and Morris water maze tasks with aversive stimuli for conditioning. Furthermore, the TLCN mutant mice showed an increase of prepulse inhibition of the acoustic startle response. These results suggest that TLCN is a determinant of the dynamic range of synaptic plasticity and plays roles in reward‐motivated learning and memory and sensorimotor gating.

Involvement of NMDAR2A tyrosine phosphorylation in depression‐related behaviour

Major depressive and bipolar disorders are serious illnesses that affect millions of people. Growing evidence implicates glutamate signalling in depression, though the molecular mechanism by which glutamate signalling regulates depression‐related behaviour remains unknown. In this study, we provide evidence suggesting that tyrosine phosphorylation of the NMDA receptor, an ionotropic glutamate receptor, contributes to depression‐related behaviour. The NR2A subunit of the NMDA receptor is tyrosine‐phosphorylated, with Tyr 1325 as its one of the major phosphorylation site. We have generated mice expressing mutant NR2A with a Tyr‐1325‐Phe mutation to prevent the phosphorylation of this site in vivo. The homozygous knock‐in mice show antidepressant‐like behaviour in the tail suspension test and in the forced swim test. In the striatum of the knock‐in mice, DARPP‐32 phosphorylation at Thr 34, which is important for the regulation of depression‐related behaviour, is increased. We also show that the Tyr 1325 phosphorylation site is required for Src‐induced potentiation of the NMDA receptor channel in the striatum. These data argue that Tyr 1325 phosphorylation regulates NMDA receptor channel properties and the NMDA receptor‐mediated downstream signalling to modulate depression‐related behaviour.

Roles of the glutamate receptor ε2 and δ2 subunits in the potentiation and prepulse inhibition of the acoustic startle reflex

We examined the regulation of the acoustic startle response in mutant mice of the N‐methyl‐d‐aspartate (NMDA)‐ and δ‐subtypes of the glutamate receptor (GluR) channel, which play important roles in neural plasticity in the forebrain and the cerebellum, respectively. Heterozygous mutant mice with reduced GluRε2 subunits of the NMDA receptor showed strongly enhanced startle responses to acoustic stimuli. On the other hand, heterozygous and homozygous mutation of the other NMDA receptor GluRε subunits exerted no, or only small effects on acoustic startle responses. The threshold of the auditory brainstem response of the GluRε2‐mutant mice was comparable to that of the wild‐type littermates. The primary circuit of the acoustic startle response is a relatively simple oligosynaptic pathway located in the lower brainstem, whilst the expression of GluRε2 is restricted to the forebrain. We thus suggest that the NMDA receptor GluRε2 subunit plays a role in the regulation of the startle reflex. Ablation of the cerebellar Purkinje cell‐specific δ2 subunit of the GluR channel exerted little effect on the acoustic startle response but resulted in the enhancement of prepulse inhibition of the reflex. Because inhibition of the acoustic startle response by a weak prepulse is a measure of sensorimotor gating, the process by which an organism filters sensory information, these observations indicate the involvement of the cerebellum in the modulation of sensorimotor gating.

Involvement of protein-tyrosine phosphatase PTPMEG in motor learning and cerebellar long-term depression

Although protein‐tyrosine phosphorylation is important for hippocampus‐dependent learning, its role in cerebellum‐dependent learning remains unclear. We previously found that PTPMEG, a cytoplasmic protein‐tyrosine phosphatase expressed in Purkinje cells (PCs), bound to the carboxyl‐terminus of the glutamate receptor δ2 via the postsynaptic density‐95/discs‐large/ZO‐1 domain of PTPMEG. In the present study, we generated PTPMEG‐knockout (KO) mice, and addressed whether PTPMEG is involved in cerebellar plasticity and cerebellum‐dependent learning. The structure of the cerebellum in PTPMEG‐KO mice appeared grossly normal. However, we found that PTPMEG‐KO mice showed severe impairment in the accelerated rotarod test. These mice also exhibited impairment in rapid acquisition of the cerebellum‐dependent delay eyeblink conditioning, in which conditioned stimulus (450‐ms tone) and unconditioned stimulus (100‐ms periorbital electrical shock) were co‐terminated. Moreover, long‐term depression at parallel fiber–PC synapses was significantly attenuated in these mice. Developmental elimination of surplus climbing fibers and the physiological properties of excitatory synaptic inputs to PCs appeared normal in PTPMEG‐KO mice. These results suggest that tyrosine dephosphorylation events regulated by PTPMEG are important for both motor learning and cerebellar synaptic plasticity.

NMDAR2B tyrosine phosphorylation regulates anxiety-like behavior and CRF expression in the amygdala

BackgroundAnxiety disorders are a highly prevalent and disabling class of psychiatric disorders. There is growing evidence implicating the glutamate system in the pathophysiology and treatment of anxiety disorders, though the molecular mechanism by which the glutamate system regulates anxiety-like behavior remains unclear.ResultsIn this study, we provide evidence suggesting that tyrosine phosphorylation of the NMDA receptor, an ionotropic glutamate receptor, contributes to anxiety-like behavior. The GluN2B subunit of the NMDA receptor is tyrosine-phosphorylated: Tyr-1472 is the major phosphorylation site. Homozygous knock-in mice that express a Tyr-1472-Phe mutant of GluN2B, which prevents phosphorylation of this site, show enhanced anxiety-like behavior in the elevated plus-maze test. Expression of corticotropin-releasing factor (CRF), which is important for the regulation of anxiety-like behavior, is increased in the amygdala of the knock-in mice. Furthermore, injection of CRF receptor antagonist attenuated the enhanced anxiety-like behavior of the knock-in mice. We also show that elevated plus-maze exposure simultaneously induced de-phosphorylation of Tyr-1472 and increased CRF expression.ConclusionsThese data suggest that Tyr-1472 phosphorylation on GluN2B is important for anxiety-like behavior by negative regulation of CRF expression in the amygdala.

Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders

Intracellular trafficking of receptor proteins is essential for neurons to detect various extracellular factors during the formation and refinement of neural circuits. However, the precise mechanisms underlying the trafficking of neurotrophin receptors to synapses remain elusive. Here, we demonstrate that a brain-enriched sorting nexin, ARHGAP33, is a new type of regulator for the intracellular trafficking of TrkB, a high-affinity receptor for brain-derived neurotrophic factor. ARHGAP33 knockout (KO) mice exhibit reduced expression of synaptic TrkB, impaired spine development and neuropsychiatric disorder-related behavioural abnormalities. These deficits are rescued by specific pharmacological enhancement of TrkB signalling in ARHGAP33 KO mice. Mechanistically, ARHGAP33 interacts with SORT1 to cooperatively regulate TrkB trafficking. Human ARHGAP33 is associated with brain phenotypes and reduced SORT1 expression is found in patients with schizophrenia. We propose that ARHGAP33/SORT1-mediated TrkB trafficking is essential for synapse development and that the dysfunction of this mechanism may be a new molecular pathology of neuropsychiatric disorders.

LMTK3 Deficiency Causes Pronounced Locomotor Hyperactivity and Impairs Endocytic Trafficking

LMTK3 belongs to the LMTK family of protein kinases that are predominantly expressed in the brain. Physiological functions of LMTK3 and other members of the LMTK family in the CNS remain unknown. In this study, we performed a battery of behavioral analyses using Lmtk3−/− mice and showed that these mice exhibit abnormal behaviors, including pronounced locomotor hyperactivity, reduced anxiety behavior, and decreased depression-like behavior. Concurrently, the dopamine metabolite levels and dopamine turnover rate are increased in the striata of Lmtk3−/− mice compared with wild-type controls. In addition, using cultured primary neurons from Lmtk3−/− mice, we found that LMTK3 is involved in the endocytic trafficking of N-methyl-d-aspartate receptors, a type of ionotropic glutamate receptor. Altered membrane traffic of the receptor in Lmtk3−/− neurons may underlie behavioral abnormalities in the mutant animals. Together, our data suggest that LMTK3 plays an important role in regulating locomotor behavior in mice.