Michisuke Yuzaki

TARGETED DISRUPTION OF NMDA RECEPTOR 1 GENE ABOLISHES NMDA RESPONSE AND RESULTS IN NEONATAL DEATH

In vitro studies have suggested that the NMDA receptor consists of an essential subunit, NR1, and various modulatory NR2 subunits. To test this hypothesis directly in vivo, we generated mice carrying a disrupted NR1 allele. NMDA-inducible increases in intracellular calcium and membrane currents were abolished in neurons from homozygous null mutants (NR1-/-). Thus, NR1 has a unique role, which cannot be substituted by any other subunit, in determining the activity of the endogenous NMDA receptor. A concomitant reduction in levels of NR2B but not NR2A occurred in NR1-/- mice, demonstrating that there is an interdependence of subunit expression. NR1-/- mice died 8-15 hr after birth, indicating a vital neonatal function for the NMDA receptor. Although the NMDA receptor has been implicated in several aspects of neurodevelopment, overall neuroanatomy of NR1-/- mice appeared normal. Pathological evidence suggested that respiratory failure was the ultimate cause of death.

Therapeutic potential of appropriately evaluated safe-induced pluripotent stem cells for spinal cord injury

Various types of induced pluripotent stem (iPS) cells have been established by different methods, and each type exhibits different biological properties. Before iPS cell-based clinical applications can be initiated, detailed evaluations of the cells, including their differentiation potentials and tumorigenic activities in different contexts, should be investigated to establish their safety and effectiveness for cell transplantation therapies. Here we show the directed neural differentiation of murine iPS cells and examine their therapeutic potential in a mouse spinal cord injury (SCI) model. “Safe” iPS-derived neurospheres, which had been pre-evaluated as nontumorigenic by their transplantation into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse brain, produced electrophysiologically functional neurons, astrocytes, and oligodendrocytes in vitro. Furthermore, when the safe iPS-derived neurospheres were transplanted into the spinal cord 9 d after contusive injury, they differentiated into all three neural lineages without forming teratomas or other tumors. They also participated in remyelination and induced the axonal regrowth of host 5HT+ serotonergic fibers, promoting locomotor function recovery. However, the transplantation of iPS-derived neurospheres pre-evaluated as “unsafe” showed robust teratoma formation and sudden locomotor functional loss after functional recovery in the SCI model. These findings suggest that pre-evaluated safe iPS clone-derived neural stem/progenitor cells may be a promising cell source for transplantation therapy for SCI.

Cbln1 is essential for synaptic integrity and plasticity in the cerebellum

Cbln1 is a cerebellum-specific protein of previously unknown function that is structurally related to the C1q and tumor necrosis factor families of proteins. We show that Cbln1 is a glycoprotein secreted from cerebellar granule cells that is essential for three processes in cerebellar Purkinje cells: the matching and maintenance of pre- and postsynaptic elements at parallel fiber–Purkinje cell synapses, the establishment of the proper pattern of climbing fiber–Purkinje cell innervation, and induction of long-term depression at parallel fiber–Purkinje cell synapses. Notably, the phenotype of cbln1-null mice mimics loss-of-function mutations in the orphan glutamate receptor, GluRδ2, a gene selectively expressed in Purkinje neurons. Therefore, Cbln1 secreted from presynaptic granule cells may be a component of a transneuronal signaling pathway that controls synaptic structure and plasticity.

Cbln1 Is a Ligand for an Orphan Glutamate Receptor δ2, a Bidirectional Synapse Organizer

Orphan No More The glutamate receptor δ2 (GluD2), another member of the ionotropic glutamate receptor family, has long been considered to be an orphan receptor because there are no known endogenous ligands. Nevertheless, GluD2 is essential for the normal development of cerebellar circuits. Using immunocytochemistry, binding assays, electrophysiology, and freeze-fracture electron microscopy, Matsuda et al. (p. 363) found that Cbln1, a soluble protein secreted from cerebellar granule cells, binds to the extracellular N terminus of GluD2 on Purkinje cells. Binding has two independent consequences: First, it leads to presynaptic differentiation and second, it causes postsynaptic clustering of several important synapse-specific molecules. Both events are needed for synapse formation between granule cells and Purkinje cells. A signaling complex serves as a synapse organizer that acts bidirectionally on both pre- and postsynaptic components. Cbln1, secreted from cerebellar granule cells, and the orphan glutamate receptor δ2 (GluD2), expressed by Purkinje cells, are essential for synapse integrity between these neurons in adult mice. Nevertheless, no endogenous binding partners for these molecules have been identified. We found that Cbln1 binds directly to the N-terminal domain of GluD2. GluD2 expression by postsynaptic cells, combined with exogenously applied Cbln1, was necessary and sufficient to induce new synapses in vitro and in the adult cerebellum in vivo. Further, beads coated with recombinant Cbln1 directly induced presynaptic differentiation and indirectly caused clustering of postsynaptic molecules via GluD2. These results indicate that the Cbln1-GluD2 complex is a unique synapse organizer that acts bidirectionally on both pre- and postsynaptic components.

Mutation of a glutamate receptor motif reveals its role in gating and δ2 receptor channel properties

Despite its importance in the cerebellum, the functions of the orphan glutamate receptor δ2 are unknown. We examined a mutant δ2 receptor channel in lurcher mice that was constitutively active in the absence of ligand. Because this mutation was within a highly conserved motif (YTANLAAF), we tested its effect on several glutamate receptors. Mutant δ2 receptors showed distinct channel properties, including double rectification of the current–voltage relationship, sensitivity to a polyamine antagonist and moderate Ca2+ permeability, whereas other constitutively active mutant glutamate channels resembled wild-type channels in these respects. Moreover, the kinetics of ligand-activated currents were strikingly altered. We conclude that the δ2 receptor has a functional ion channel pore similar to that of glutamate receptors. The motif may have a role in the channel gating of glutamate receptors.

Cloning and characterization of a novel NMDA receptor subunit NR3B: A dominant subunit that reduces calcium permeability

We report the cloning and characterization of a novel NMDA receptor subunit cDNA, which encodes a predicted polypeptide of 1003 amino acids. Phylogenic analysis indicates that this new subunit is most closely related to NR3A. Therefore, we term it NR3B. Important functional domains of glutamate receptors, such as the ligand-binding domain, the channel pore, and the channel gate, are conserved in NR3B. NR3B mRNA was expressed highly in pons, midbrain, medulla, and the spinal cord, but at low levels in the forebrain and the cerebellum. Although NR3A mRNA expression decreases sharply after the second postnatal weeks, NR3B mRNA expression levels in whole brain were constant during postnatal development and into adult. Coimmunoprecipitation analysis showed that NR3B could form NMDA receptor complex with NR1a and NR2A subunits in heterologous cells. Although expression of NR3B alone did not reconstitute functional NMDA receptors, coexpression of NR3B reduced the Ca(2+) permeability of glutamate-induced currents in cells expressing NR1a and NR2A. These results indicate that NR3B is a dominant modulatory subunit that can modify the function of NMDA receptors. Since high Ca(2+) permeability of NMDA receptors is thought to be a key feature for NMDA receptors to play critical roles in neurodevelopment, synaptic plasticity, and neuronal death, NR3B may contribute to the regulation of these physiological and pathological processes.

Pharmacological and immunocytochemical characterization of metabotropic glutamate receptors in cultured Purkinje cells

Metabotropic glutamate receptor (mGluR) is highly expressed in cerebellar Purkinje cells. The purpose of this study was pharmacological and immunocytochemical characterization of the mGluR in single cerebellar neurons, especially Purkinje cells. Ca2+ imaging with fura-2 in cultured cerebellar neurons, identified immunocytochemically, was used to record the direct effects of drugs in stable conditions. In addition, the expression of mGluR was examined, and expression of the intracellular receptor for inositol trisphosphate (IP3) produced by mGluR activation was studied immunocytochemically with specific antibodies. Purkinje neurons and some other neurons showed Ca(2+)- mobilizing responses to mGluR agonists. These responses were mediated by mGluR because they were not blocked by ionotropic GluR antagonists, were independent of the caffeine-sensitive Ca2+ pool, and were blocked by inhibitors of IP3-induced Ca2+ release. This is the first pharmacological characterization of mGluR at single Purkinje cells. The results differed as follows from those in earlier studies in which phosphoinositide turnover of the entire population of cerebellar cells was monitored: (1) the mGluR responses were not blocked by pertussis toxin or D,L-2-amino-3-phosphonopropionic acid; (2) glutamate was a potent agonist, whereas L-aspartate was ineffective; and (3) the dose- response relationship showed an all-or-none tendency. The metaboltropic response of Purkinje cells changed markedly during development, with a sharp peak after day 4 of culture, whereas mGluR and IP3 receptor proteins increased steadily during maturation. This apparent desensitization of mGluR was not blocked by inhibitors of protein kinase C (PKC) or ADP-ribosyltransferase. The metabotropic responses were mainly localized to the center of the somata of Purkinje cells even on day 4, whereas both receptor proteins were expressed throughout the cell. These results suggest that the function of mGluR is spatially and developmentally controlled by a posttranslational mechanism involving a mechanism other than phosphorylation by PKC or ADP- ribosylation.

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.

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.

Accumulation of AMPA Receptors in Autophagosomes in Neuronal Axons Lacking Adaptor Protein AP-4

AP-4 is a member of the adaptor protein complexes, which control vesicular trafficking of membrane proteins. Although AP-4 has been suggested to contribute to basolateral sorting in epithelial cells, its function in neurons is unknown. Here, we show that disruption of the gene encoding the beta subunit of AP-4 resulted in increased accumulation of axonal autophagosomes, which contained AMPA receptors and transmembrane AMPA receptor regulatory proteins (TARPs), in axons of hippocampal neurons and cerebellar Purkinje cells both in vitro and in vivo. AP-4 indirectly associated with the AMPA receptor via TARPs, and the specific disruption of the interaction between AP-4 and TARPs caused the mislocalization of endogenous AMPA receptors in axons of wild-type neurons. These results indicate that AP-4 may regulate proper somatodendritic-specific distribution of its cargo proteins, including AMPA receptor-TARP complexes and the autophagic pathway in neurons.