Wataru Kakegawa

Impaired cerebellar development and function in mice lacking CAPS2, a protein involved in neurotrophin release

Ca2+-dependent activator protein for secretion 2 (CAPS2/CADPS2) is a secretory granule-associated protein that is abundant at the parallel fiber terminals of granule cells in the mouse cerebellum and is involved in the release of neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF), both of which are required for cerebellar development. The human homolog gene on chromosome 7 is located within susceptibility locus 1 of autism, a disease characterized by several cerebellar morphological abnormalities. Here we report that CAPS2 knock-out mice are deficient in the release of NT-3 and BDNF, and they consequently exhibit suppressed phosphorylation of Trk receptors in the cerebellum; these mice exhibit pronounced impairments in cerebellar development and functions, including neuronal survival, differentiation and migration of postmitotic granule cells, dendritogenesis of Purkinje cells, lobulation between lobules VI and VII, structure and vesicular distribution of parallel fiber–Purkinje cell synapses, paired-pulse facilitation at parallel fiber–Purkinje cell synapses, rotarod motor coordination, and eye movement plasticity in optokinetic training. Increased granule cell death of the external granular layer was noted in lobules VI–VII and IX, in which high BDNF and NT-3 levels are specifically localized during cerebellar development. Therefore, the deficiency of CAPS2 indicates that CAPS2-mediated neurotrophin release is indispensable for normal cerebellar development and functions, including neuronal differentiation and survival, morphogenesis, synaptic function, and motor leaning/control. The possible involvement of the CAPS2 gene in the cerebellar deficits of autistic patients is discussed.

Differential Roles of Glial and Neuronal Glutamate Transporters in Purkinje Cell Synapses

Glutamate transporters are essential for terminating excitatory neurotransmission. Two distinct glutamate transporters, glutamate–aspartate transporter (GLAST) and excitatory amino acid transporter 4 (EAAT4), are expressed most abundantly in the molecular layer of the cerebellar cortex. GLAST is expressed in Bergmann glial processes surrounding excitatory synapses on Purkinje cell dendritic spines, whereas EAAT4 is concentrated on the extrasynaptic regions of Purkinje cell spine membranes. To clarify the functional significance of the coexistence of these transporters, we analyzed the kinetics of EPSCs in Purkinje cells of mice lacking either GLAST or EAAT4. There was no difference in the amplitude or the kinetics of the rising and initial decay phase of EPSCs evoked by stimulations of climbing fibers and parallel fibers between wild-type and EAAT4-deficient mice. However, long-lasting tail currents of the EPSCs appeared age dependently in most of Purkinje cells in EAAT4-deficient mice. These tail currents were never seen in mice lacking GLAST. In the GLAST-deficient mice, however, the application of cyclothiazide that reduces desensitization of AMPA receptors increased the peak amplitude of the EPSC and prolonged its decay more markedly than in both wild-type and EAAT4-deficient mice. The results indicate that these transporters play differential roles in the removal of synaptically released glutamate. GLAST contributes mainly to uptake of glutamate that floods out of the synaptic cleft at early times after transmitter release. In contrast, the main role of EAAT4 is to remove low concentrations of glutamate that escape from the uptake by glial transporters at late times and thus prevents the transmitter from spilling over to neighboring synapses.

Rescue of abnormal phenotypes of the δ2 glutamate receptor-null mice by mutant δ2 transgenes

The δ2 glutamate receptor (GluRδ2) has a crucial role in cerebellar functions; disruption of GluRδ2 alleles in mice (δ2−/−) impairs synapse formation and long‐term depression, which is thought to underlie motor learning in the cerebellum, and consequently leads to motor discoordination. However, it has been unclear whether GluRδ2 is activated by glutamate analogues. Here we introduced a GluRδ2 transgene, which had a mutation (Arg514Lys) in the putative ligand‐binding motif conserved in all mammalian ionotropic glutamate receptors (iGluRs) and their ancestral bacterial periplasmic amino‐acid‐binding proteins, into δ2−/− mice. Surprisingly, a mutant GluRδ2 transgene, as well as a wild‐type GluRδ2 transgene, rescued all abnormal phenotypes of δ2−/− mice. Therefore, these results indicate that the conserved arginine residue, which is crucial for the binding of iGluRs to glutamate analogues, is not essential for the restoration of GluRδ2 functions in δ2−/− mice.

Sindbis viral-mediated expression of Ca2+-permeable AMPA receptors at hippocampal CA1 synapses and induction of NMDA receptor-independent long-term potentiation.

Gene manipulation in order to artificially express a particular gene in neurons in the central nervous system is a powerful tool for the analysis of brain function. Sindbis viral vectors have been developed to express high levels of foreign genes in postmitotic brain neurons with little transfection of glial cells. In this study, we expressed the gene encoding the unedited GluR2 (GluR‐B) subunit of the AMPA‐type glutamate receptor that forms inwardly rectifying and Ca2+‐permeable channels, in rat CA1 hippocampal neurons in slice cultures using Sindbis viral vectors. The pyramidal cell layer of the CA1 region was injected with recombinant Sindbis viruses encoding both enhanced green fluorescent protein (GFP) and unedited GluR2. The GFP fluorescence from CA1 neurons could be detected as early as 6u2003h and reached a maximal level about 48u2003h postinfection. The inwardly rectifying and Ca2+‐permeable AMPA receptors were expressed in most CA1 pyramidal cells expressing GFP. These AMPA receptors expressed by gene transfer were involved in fast excitatory neurotransmission elicited by electrical stimulation of the Schaffer collaterals in the stratum radiatum. Tetanic stimulation of Schaffer collaterals induced NMDA receptor‐independent, long‐term potentiation due to Ca2+ influx through the newly expressed AMPA receptors in the area densely stained with GFP. Thus, the combined use of Sindbis viral vectors with the GFP reporter allowed physiological examination of the roles of a specific gene product in synaptic function in well‐characterized brain neurons.

ERK1/2 but not p38 MAP kinase is essential for the long-term depression in mouse cerebellar slices

Mitogen‐activated protein kinase (MAPK) cascade is essential for synaptic plasticity and learning. In the hippocampus, three different MAPK subfamilies, extracellular signal‐regulated kinaseu20031/2 (ERK1/2), p38 MAPK and c‐Jun NH2‐terminal protein kinase (JNK), selectively regulate activity‐dependent glutamate receptor trafficking during long‐term potentiation (LTP), long‐term depression (LTD), and depotentiation after LTP, respectively. Although LTP and LTD at cerebellar parallel fibre (PF)–Purkinje cell synapses are thought to be controlled by glutamate receptor trafficking, the involvement of MAPK subfamilies has not been systemically studied in cerebellar slice preparations. To clarify the role of the MAPK cascade in cerebellar LTD, we performed biochemical and electrophysiological analyses using ICR mouse cerebellar slices. Immunoblot analyses using phosphorylation‐specific antibodies for MAPKs revealed that among the three MAPKs, ERK1/2 was specifically activated by phorbol ester, which could induce LTD in cerebellar slices. In addition, U0126, a specific inhibitor of the MAPK kinase‐ERK1/2 pathway, abrogated the induction of LTD in cerebellar slices, whereas SB203580 and SP600125, specific inhibitors of p38 MAPK and JNK, respectively, had no effect. Although metabotropic glutamate receptoru20031 (mGluR1) has been suggested as a possible downstream target of ERK1/2 in cell‐culture preparations, mGluR1‐activated slow excitatory postsynaptic currents (EPSCs) were not affected by U0126 treatment in slices. These findings indicate that unlike hippocampal LTD mediated by p38 MAPK, glutamate receptor trafficking during cerebellar LTD was regulated by a distinct mechanism involving ERK1/2 in slice preparations.

Induction of long‐term depression and phosphorylation of the δ2 glutamate receptor by protein kinase C in cerebellar slices

The phosphorylation of ionotropic glutamate receptors (iGluRs) by protein kinases plays a crucial role in synaptic plasticity. In the cerebellum, protein kinase C (PKC) activation is required for the induction of long‐term depression (LTD) at parallel fibre–Purkinje cell synapses. Although δ2 glutamate receptors (GluRδ2), expressed predominantly in Purkinje cells, are essential for cerebellar LTD, little is known about the mechanism by which GluRδ2 participates in LTD or its relationship with PKC activation pathways. We found that a PKC activator, phorbol ester, induced postsynaptic LTD in Purkinje cells in mouse cerebellar slice preparations without significantly changing the presynaptic properties. Under this condition, the GluRδ2 prepared from the cerebellar slices was significantly phosphorylated. Indeed, the C‐terminus of the GluRδ2 fused with glutathione‐S‐transferase (GST) was directly phosphorylated by purified PKC at a specific serine residue. In addition, two‐dimensional phosphopeptide mapping analysis indicated that the major phosphorylation site of the GST‐fusion protein containing the C‐terminus of GluRδ2 was identical to that of GluRδ2 prepared from cerebellar slices. Therefore, GluRδ2 is phosphorylated by PKC in vitro and by an LTD‐inducing stimulus in slice preparations. Because this region of GluRδ2 is known to associate with certain intracellular molecules, the PKC phosphorylation status of the C‐terminus of GluRδ2 may be involved in new signaling pathways during LTD.

Ho15J- : A new hotfoot allele in a hot spot in the gene encoding the δ2 glutamate receptor

Hotfoot, a recessive mouse mutation characterized by ataxia and jerky movements of the hindlimbs, is caused by various mutations in the gene (Grid2) encoding the delta2 glutamate receptor (GluRdelta2). So far, at least 20 alleles, arising either spontaneously or through the random insertion of transgenes, have been described. Interestingly, most hotfoot mutants have deletions of one or more exons coding for portions of the most amino-terminal domain of GluRdelta2. However, because live mice colonies are no longer available for most hotfoot mutants, the possibility that the loss of a part of an intron might affect the splicing of other exons or the general efficiency of transcription could not be ruled out. Here, we report that a newly identified hotfoot mutant, ho15J, was caused by an intragenic deletion of the Grid2 gene, which indeed resulted in a new type of 52-amino-acid deletion in the most amino-terminal domain of GluRdelta2. Like GluRdelta2 proteins in ho4J mutants, GluRdelta2 proteins in ho15J mice were retained in the soma of Purkinje cells, where they were degraded. Long-term depression, a form of synaptic plasticity underlying information storage in the cerebellum, was abrogated, and ho15J mice showed severe motor discoordination on rotarod tests. The agreement between the PCR results for genomic DNA and the RT-PCR results for the ho15J allele supports the view that PCR analyses of grid2 genomic DNA can predict alterations in mRNA and protein. In addition, the present findings underscore the importance of the most amino-terminal domain in GluRdelta2 signaling and cerebellar functions.

Functional NMDA receptor channels generated by NMDAR2B gene transfer in rat cerebellar Purkinje cells

The adult cerebellar Purkinje cell is an exceptional neuron in the central nervous system in that it expresses high levels of NMDAR1 (NR1) mRNA without expressing any NMDAR2 (NR2) mRNAs. It has no functional NMDA receptor (NMDAR) channels, although it receives enormous numbers of excitatory inputs. Despite the high level of NR1 mRNA expression, the presence and localization of NR1 protein in mature Purkinje cells are controversial. To examine the presence of NR1 protein and its ability to form functional NMDARs, we expressed the NR2B subunit in rat mature Purkinje neurons by Sindbis viral‐mediated gene transfer. The recombinant virus encoding both the NR2B and enhanced green fluorescent protein (GFP) genes (designated as SIN–EG–NR2B) infected Purkinje cells without infecting glial cells. GFP fluorescence was detected in the soma and throughout dendrites of Purkinje cells 18–24u2003h postinfection. In most of GFP‐positive cells, the expression of NR2B protein was detected by immunostaining with NR2B‐specific antibodies. In Purkinje cells infected with SIN–EG–NR2B, the iontophoretic application of NMDA induced prominent NMDAR‐mediated current responses, indicating that the exogenous NR2B was assembled with endogenous NR1 to form functional NMDARs. Furthermore, NMDAR‐mediated synaptic currents were detected at both the climbing fibre and parallel fibre synapses in infected Purkinje cells. Thus, the mature Purkinje cell produces NR1 protein that is ready to combine with NR2 to form functional NMDARs in excitatory synapses.