Sumiko Mikawa

Disruption of AMPA receptor GluR2 clusters following long‐term depression induction in cerebellar Purkinje neurons

Cerebellar long‐term depression (LTD) is thought to play an important role in certain types of motor learning. However, the molecular mechanisms underlying this event have not been clarified. Here, using cultured Purkinje cells, we show that stimulations inducing cerebellar LTD cause phosphorylation of Ser880 in the intracellular C‐terminal domain of the AMPA receptor subunit GluR2. This phosphorylation is accompanied by both a reduction in the affinity of GluR2 to glutamate receptor interacting protein (GRIP), a molecule known to be critical for AMPA receptor clustering, and a significant disruption of postsynaptic GluR2 clusters. Moreover, GluR2 protein released from GRIP is shown to be internalized. These results suggest that the dissociation of postsynaptic GluR2 clusters and subsequent internalization of the receptor protein, initiated by the phosphorylation of Ser880, are the mechanisms underlying the induction of cerebellar LTD.

Phosphorylation of serine-880 in GluR2 by protein kinase C prevents its C terminus from binding with glutamate receptor-interacting protein

Abstract : Phosphorylation of the glutamate receptor is an important mechanism of synaptic plasticity. Here, we show that the C terminus of GluR2 of the α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptor is phosphorylated by protein kinase C and that serine‐880 is the major phosphorylation site. This phosphorylation also occurs in human embryonic kidney (HEK) cells by addition of 12‐O‐tetradecanoylphorbol 13‐acetate. Our immunoprecipitation experiment revealed that the phosphorylation of serine‐880 in GluR2 drastically reduced the affinity for glutamate receptor‐interacting protein (GRIP), a synaptic PDZ domain‐containing protein, in vitro and in HEK cells. This result suggests that modulation of serine‐880 phosphorylation in GluR2 controls the clustering of AMPA receptors at excitatory synapses and consequently contributes to synaptic plasticity.

New role of δ2-glutamate receptors in AMPA receptor trafficking and cerebellar function

Previous gene knockout studies have shown that the orphan glutamate receptor δ2 (GluRδ2) is critically involved in synaptogenesis between parallel fibers and Purkinje cells during development. However, the precise function of GluRδ2 and whether it is functional in the mature cerebellum remain unclear. To address these issues, we developed an antibody specific for the putative ligand-binding region of GluRδ2, and application of this antibody to cultured Purkinje cells induced AMPA receptor endocytosis, attenuated synaptic transmission and abrogated long-term depression. Moreover, injection of this antibody into the subarachnoidal supracerebellar space of adult mice caused transient cerebellar dysfunction, such as ataxic gait and poor performance in the rotorod test. These results indicate that GluRδ2 is involved in AMPA receptor trafficking and cerebellar function in adult mice.

Developmental changes in KCC1, KCC2 and NKCC1 mRNAs in the rat cerebellum.

Cation chloride cotransporters are considered to play pivotal roles in controlling the intracellular and extracellular ionic environments of neurons, hence controlling neuronal function. To establish how these cotransporters are involved in cerebellum development, we investigated the expression of KCC1, KCC2 and NKCC1 mRNAs in the developing rat cerebellum using in situ hybridization histochemistry. In the external germinal layer, where premature cells exist, we found substantial KCC1 and NKCC1 mRNA expression on P7 and P14, while KCC2 mRNA was not detected. In contrast, KCC2 mRNA was already expressed in Purkinje cells on P1. We also observed KCC2 mRNA expression in postmigratory granule cells after P7. The expression of KCC1, KCC2, and NKCC1 mRNAs reached adult patterns by P21. In the adult cerebellum, KCC2 mRNA was expressed in most neurons, including Purkinje cells, granule cells, and stella/basket cells, while KCC1 and NKCC1 mRNAs were only detected in granule cells and glial cells. These findings suggest that in the rat cerebellum KCC2 mRNA expression is induced when neurons arrive their final destinations.

Glycine cleavage system in neurogenic regions.

The glycine cleavage system (GCS) is the essential enzyme complex for degrading glycine and supplying 5,10‐methylenetetrahydrofolate for DNA synthesis. Inherited deficiency of this system causes nonketotic hyperglycinemia, characterized by severe neurological symptoms and frequent association of brain malformations. Although high levels of glycine have been considered to cause the above‐mentioned problems, the detailed pathogenesis of this disease is still unknown. Here we show that GCS is abundantly expressed in rat embryonic neural stem/progenitor cells in the neuroepithelium, and this expression is transmitted to the radial glia–astrocyte lineage, with prominence in postnatal neurogenic regions. These data indicate that GCS plays important roles in neurogenesis, and suggest that disturbance of neurogenesis induced by deficiency of GCS may be the main pathogenesis of nonketotic hyperglycinemia.

Imaging of phosphatidylcholines in the adult rat brain using MALDI-TOF MS.

Phosphatidylcholines (PCs) are the most abundant constituents of lipid in the brain. PCs function as major structural components of cell membranes and as important sources for signaling molecules. In the brain, three kinds of PCs, dipalmitoyl PC, palmitoyloleoyl PC, and stearoyloleoyl PC have been reported to be major species. They have different chemical and biological characteristics depending on the length of alkyl chains and the degree of saturation, suggesting that the abundance of PCs might be important to keep specialized membrane structures in the brain, such as myelin and synaptic membranes. However, detailed imaging of PCs in the total rat brain has not done yet. Thus, using imaging technology by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), we investigated the total distribution of PC32:0, PC34:1, and PC36:1 in the rat brain. PC32:0 and PC34:1 were more abundantly observed in the gray matter areas than in the white matter areas throughout the central nervous system (CNS), while PC36:1 was evenly seen at low levels in both areas. In addition, we found that PC32:0 and PC34:1 were detected at very high levels in the granular layer of the olfactory bulb, piriform cortex, insular cortex, and molecular layer of the cerebellum, which are known for areas showing high neuronal plasticity. The present imaging data clearly show that various PCs are differentially distributed throughout the rat CNS, and suggest that these differential distributions of various PCs are necessary to keep normal brain functions.

Irradiation in Adulthood as a New Model of Schizophrenia

Background Epidemiological studies suggest that radiation exposure may be a potential risk factor for schizophrenia in adult humans. Here, we investigated whether adult irradiation in rats caused behavioral abnormalities relevant to schizophrenia. Methodology/Principal Findings A total dose of 15-Gy irradiation in six fractionations during 3 weeks was exposed to the forebrain including the subventricular zone (SVZ) and subgranular zone (SGZ) with male rats in the prone position. Behavioral, immunohistochemical, and neurochemical studies were performed three months after fractionated ionizing irradiation. Three months after fractionated ionizing irradiation, the total numbers of BrdU-positive cells in both the SVZ and SGZ zones of irradiated rats were significantly lower than those of control (sham-irradiated) rats. Hyperactivity after administration of the dopaminergic agonist methamphetamine, but not the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine, was significantly enhanced in the irradiated rats although spontaneous locomotion in the irradiated rats was significantly lower than that of controls. Behavioral abnormalities including auditory sensory gating deficits, social interaction deficits, and working memory deficits were observed in the irradiated rats. Conclusion/Significance The present study suggests that irradiation in adulthood caused behavioral abnormalities relevant to schizophrenia, and that reduction of adult neurogenesis by irradiation may be associated with schizophrenia-like behaviors in rats.

Bone morphogenetic protein-4 expression in the adult rat brain.

Bone morphogenetic protein‐4 (BMP4) is a member of the transforming growth factor‐β (TGF‐β) superfamily and plays important roles in multiple biological events. Although BMP4 expression has been well described in the early development of the central nervous system (CNS), little information is available on its expression in the adult CNS. Therefore, we investigated BMP4 expression in the adult rat CNS by using immunohistochemistry. BMP4 is intensely expressed in most neurons and their dendrites. In addition, intense BMP4 expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. Furthermore, we found that astrocytes also express BMP4 protein. These data indicate that BMP4 is more widely expressed throughout the adult CNS than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that BMP4 plays pivotal roles also in the adult brain. J. Comp. Neurol. 499:613–625, 2006.

BMP2 expression in the adult rat brain

Bone morphogenetic protein‐2 (BMP2) is a member of the transforming growth factor‐β (TGF‐β) superfamily and plays important roles in multiple biological events. Although BMP2 expression has been well described in the early development of the central nervous system (CNS), little information is available on its expression in the adult CNS. We thus investigated BMP2 expression in the adult rat CNS by using immunohistochemistry. Here we show that BMP2 is widely expressed throughout the adult CNS. In addition, besides intense BMP2 expression in almost all neurons, we found BMP2 expression in astrocytes and ependymal cells. Interestingly, we found that the axons of olfactory sensory neurons express BMP2. In addition, in the glomerular layer, BMP2 was very strongly expressed in some glomeruli, whereas the other glomeruli were weakly stained, suggesting that the variations in BMP2 expression level in each glomerus might be cues for each axon to find its adequate target and to keep its identity. Furthermore, we compared the expression patterns of BMP2 and BMP4. Interestingly, BMP4 was preferentially expressed in the dendrites of several neurons, whereas BMP2 was basically not expressed in the dendrites; however, it was detected in the axons. This means that in a single neuron the localizations of BMP2 and BMP4 are differentially regulated. These data indicate that BMP2 is more widely expressed throughout the adult CNS than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that BMP2 also plays pivotal roles in the adult brain. J. Comp. Neurol. 518:4513–4530, 2010.

Bone morphogenetic protein receptor expressions in the adult rat brain.

Bone morphogenetic proteins (BMP) are members of the transforming growth factor β (TGF-β) superfamily. BMPs exert its biological functions by interacting with membrane bound receptors belonging to the serine/threonine kinase family including bone morphogenetic protein receptor I (BMPRIA, BMPRIB) and type II (BMPRII). Although BMPR expressions have been well described in the early development of the CNS, little information is available for their expressions in the adult CNS. We, thus, investigated BMPR expressions in the adult rat CNS using immunohistochemistry. Here, we show that BMPRIA, IB and II proteins are widely expressed throughout the adult CNS. Interestingly, we observed that BMPRIA, IB and II proteins are abundantly expressed in many kinds of axons. In addition, we found that BAMRIB-IR was preferentially expressed in dendrites of many neurons throughout the CNS, while BMPRIA was mainly expressed in cell bodies, showing that BMPRIA and BMPRIB are differentially targeted in a single neuron. In addition, besides abundant BMPR expressions in neurons, we exhibited BMPR expressions in astrocytes and ependymal cells. These data indicate that BMPRs are more widely expressed throughout the adult CNS than previously reported, and their continued abundant expressions in the adult brain strongly support the idea that BMPRs play pivotal roles also in the adult brain.