Cyclin-dependent kinase 5 is required for normal cerebellar development.

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase, and its kinase activity is dependent upon its association with either of the activating subunits p35 or p39, which are mainly expressed in neurons. We previously reported that Cdk5 knockout (KO) mice exhibit perinatal lethality, defective neuronal migration, and abnormal positioning of neurons in the facial motor nucleus and inferior olive in the hindbrain and Purkinje cells (PCs) in the cerebellum. In this study, we focused on the analysis of the role of Cdk5 in cerebellar development. For this purpose we generated midbrain-hindbrain-specific Cdk5 conditional knockout (MHB-Cdk5 KO) mice because the cerebellum develops postnatally, whereas Cdk5 KO mice die perinatally. Histological analysis of the MHB-Cdk5 KO mice revealed a significant size reduction of the cerebellum. In addition, profound disturbance of inward migration of granule cells (GC) was observed in the developing cerebellum. A normal dendritic development of the Purkinje cells (PCs) was disturbed in MHB-Cdk5 KO mice. Cultured Cdk5-null PCs showed similar dendritic abnormalities. These results indicate that Cdk5/p35 plays an important role in neuronal migration of PCs and GCs and dendrite formation of PCs in cerebellar development.

Localization of inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae.

Activation of various receptors by extracellular ligands induces an influx of Ca2+ through the plasma membrane, but its molecular mechanism remains elusive and seems variable in different cell types. In the present study, we utilized mAbs generated against the cerebellar type I inositol 1,4,5-trisphosphate (InsP3) receptor and performed immunocytochemical and immunochemical experiments to examine its localization in several non-neuronal cells. By immunogold electron microscopy of ultrathin frozen sections as well as permeabilized tissue specimens, we found that a mAb to the type I InsP3 receptor (mAb 4C11) labels the plasma membrane of the endothelium, smooth muscle cell and keratinocyte in vivo. Interestingly, the labeling with the antibody was confined to caveolae, smooth vesicular inpocketings of the plasma membrane. The reactive protein, with an M(r) of 240,000 by SDS-PAGE, could be biotinylated with a membrane-impermeable reagent, sulfo-NHS-biotin, in intact cultured endothelial cells, and recovered by streptavidin-agarose beads, which result further confirmed its presence on the cell surface. The present findings indicate that a protein structurally homologous to the type I InsP3 receptor is localized in the caveolar structure of the plasma membrane and might be involved in the Ca2+ influx.

Calcium waves along the cleavage furrows in cleavage-stage Xenopus embryos and its inhibition by heparin.

Calcium signaling is known to be associated with cytokinesis; however, the detailed spatio-temporal pattern of calcium dynamics has remained unclear. We have studied changes of intracellular free calcium in cleavage-stage Xenopus embryos using fluorescent calcium indicator dyes, mainly Calcium Green-1. Cleavage formation was followed by calcium transients that localized to cleavage furrows and propagated along the furrows as calcium waves. The calcium transients at the cleavage furrows were observed at each cleavage furrow at least until blastula stage. The velocity of the calcium waves at the first cleavage furrow was approximately 3 microns/s, which was much slower than that associated with fertilization/egg activation. These calcium waves traveled only along the cleavage furrows and not in the direction orthogonal to the furrows. These observations imply that there exists an intracellular calcium-releasing activity specifically associated with cleavage furrows. The calcium waves occurred in the absence of extracellular calcium and were inhibited in embryos injected with heparin an inositol 1,4,5-trisphosphate (InsP3) receptor antagonist. These results suggest that InsP3 receptor-mediated calcium mobilization plays an essential role in calcium wave formation at the cleavage furrows.

Membrane topogenesis of a type I signal-anchor protein, mouse synaptotagmin II, on the endoplasmic reticulum.

Synaptotagmin II is a type I signal-anchor protein, in which the NH(2)-terminal domain of 60 residues (N-domain) is located within the lumenal space of the membrane and the following hydrophobic region (H-region) shows transmembrane topology. We explored the early steps of cotranslational integration of this molecule on the endoplasmic reticulum membrane and demonstrated the following: (a) The translocation of the N-domain occurs immediately after the H-region and the successive positively charged residues emerge from the ribosome. (b) Positively charged residues that follow the H-region are essential for maintaining the correct topology. (c) It is possible to dissect the lengths of the nascent polypeptide chains which are required for ER targeting of the ribosome and for translocation of the N-domain, thereby demonstrating that different nascent polypeptide chain lengths are required for membrane targeting and N-domain translocation. (d) The H-region is sufficiently long for membrane integration. (e) Proline residues preceding H-region are critical for N-domain translocation, but not for ER targeting. The proline can be replaced with amino acid with low helical propensity.

Apical vesicles bearing inositol 1,4,5-trisphosphate receptors in the Ca2+ initiation site of ductal epithelium of submandibular gland.

In polarized epithelial cells, agonists trigger Ca2+ waves and oscillations. These patterns may be caused by the compartmentalization of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools into specific regions. We have investigated the relationship between the distribution of IP3 receptors (IP3Rs) and the spatiotemporal pattern of Ca2+ signaling in the duct cells of the rat submandibular gland (SMG). Using immunofluorescence, although labeling was somewhat heterogeneous, the IP3Rs were colocalized to the apical pole of the duct cells. Immunoelectron microscopy identified small apical vesicles bearing IP3R2 in some types of duct cells. Real-time confocal imaging of intact ducts demonstrated that, after carbachol stimulation, an initial Ca2+ spike occurred in the apical region. Subsequently, repetitive Ca2+ spikes spread from the apical to the middle cytoplasm. These apical Ca2+ initiation sites were found only in some "pioneer cells," rather than in all duct cells. We performed both Ca2+ imaging and immunofluorescence on the same ducts and detected the strongest immunosignals of IP3R2 in the Ca2+ initiation sites of the pioneer cells. The subcellular localization and expression level of IP3Rs correlated strongly with the spatiotemporal nature of the intracellular Ca2+ signal and distinct Ca2+ responses among the rat SMG duct cells.

Differentiation of a teratocarcinoma line: preferential development of cholinergic neurons.

A line of embryonal carcinoma cells, PCC7-S, established in vitro from a spontaneous testicular teratocarcinoma, has been studied. Upon removing the cells from a low density monolayer culture system and permitting the cells to form aggregates in suspension, we observed a change of several physical and biochemical parameters: (a) reduction in average cell volume, (b) blockage and accumulation of cells in G1, (c) rise in secreted protease activity, (d) rise in acetylcholinesterase and choline acetyltransferase activities, and (e) disappearance of embryonic antigen F9. Although PCC7 aggregates did not undergo substantial morphological changes while suspended, when aggregates 4 or more days old were allowed to attach to plastic tissue culture dishes, substantial neurite outgrowth occurred over the next 1-3 d. This process was markedly enhanced by the addition to the growth medium of carboxymethylcellulose and inhibitors of DNA synthesis. Transmission electron microscopy disclosed a neurite ultrastructure consistent with that of neuronal processes. A veratridine-stimulated, tetrodotoxin-blocked sodium influx of 100 nmol/min per mg protein was also observed in these differentiated surface cultures. This cell line is discussed in terms of its utility for the study of early events leading to a commitment to cellular differentiation, as well as for the investigation of terminal differentiation to cholinergic neurons.

Block of Ca2+ wave and Ca2+ oscillation by antibody to the inositol 1,4,5-trisphosphate receptor in fertilized hamster eggs.

The concentration of cytoplasmic free calcium (Ca2+) increases in various stimulated cells in a wave (Ca2+ wave) and in periodic transients (Ca2+ oscillations). These phenomena are explained by inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release (IICR) and Ca(2+)-induced Ca2+ release (CICR) from separate intracellular stores, but decisive evidence is lacking. A monoclonal antibody to the IP3 receptor inhibited both IICR and CICR upon injection of IP3 and Ca2+ into hamster eggs, respectively. The antibody completely blocked sperm-induced Ca2+ waves and Ca2+ oscillations. The results indicate that Ca2+ release in fertilized hamster eggs is mediated solely by the IP3 receptor, and Ca(2+)-sensitized IICR, but not CICR, generates Ca2+ waves and Ca2+ oscillations.

Role of inositol 1,4,5-trisphosphate receptor in ventral signaling in Xenopus embryos.

The inositol 1,4,5-trisphosphate (IP3) receptor is a calcium ion channel involved in the release of free Ca2+ from intracellular stores. For analysis of the role of IP3-induced Ca2+ release (IICR) on patterning of the embryonic body, monoclonal antibodies that inhibit IICR were produced. Injection of these blocking antibodies into the ventral part of early Xenopus embryos induced modest dorsal differentiation. A close correlation between IICR blocking potencies and ectopic dorsal axis induction frequency suggests that an active IP3-Ca2+ signal may participate in the modulation of ventral differentiation.

Regulation of nerve growth mediated by inositol 1,4,5-trisphosphate receptors in growth cones.

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) acts as a Ca2+ release channel on internal Ca2+ stores. Type 1 IP3R (IP3R1) is enriched in growth cones of neurons in chick dorsal root ganglia. Depletion of internal Ca2+ stores and inhibition of IP3 signaling with drugs inhibited neurite extension. Microinjection of heparin, a competitive IP3R blocker, induced neurite retraction. Acute localized loss of function of IP3R1 in the growth cone induced by chromophore-assisted laser inactivation resulted in growth arrest and neurite retraction. IP3-induced Ca2+ release in growth cones appears to have a crucial role in control of nerve growth.

Functional expression of a mammalian odorant receptor.

Candidate mammalian odorant receptors were first cloned some 6 years ago. The physiological function of these receptors in initiating transduction in olfactory receptor neurons remains to be established. Here, a recombinant adenovirus was used to drive expression of a particular receptor gene in an increased number of sensory neurons in the rat olfactory epithelium. Electrophysiological recording showed that increased expression of a single gene led to greater sensitivity to a small subset of odorants.

Activation of the G protein Gq/11 through tyrosine phosphorylation of the alpha subunit.

Various receptors coupled to the heterotrimeric guanine nucleotide-binding protein Gq/11 stimulate formation of inositol-1,4,5-trisphosphate (IP3). Activation of these receptors also induces protein tyrosine phosphorylation. Formation of IP3 in response to stimulated receptors that couple to Gq/11 was blocked by protein tyrosine kinase inhibitors. These inhibitors appeared to act before activation of Gq/11. Moreover, stimulation of receptors coupled to Gq/11 induced phosphorylation on a tyrosine residue (Tyr356) of the Galphaq/11 subunit, and this tyrosine phosphorylation event was essential for Gq/11 activation. Tyrosine phosphorylation of Galphaq/11 induced changes in its interaction with receptors. Therefore, tyrosine phosphorylation of Galphaq/11 appears to regulate the activation of Gq/11 protein.

Requirement of the inositol trisphosphate receptor for activation of store-operated Ca2+ channels.

The coupling mechanism between endoplasmic reticulum (ER) calcium ion (Ca2+) stores and plasma membrane (PM) store-operated channels (SOCs) is crucial to Ca2+ signaling but has eluded detection. SOCs may be functionally related to the TRP family of receptor-operated channels. Direct comparison of endogenous SOCs with stably expressed TRP3 channels in human embryonic kidney (HEK293) cells revealed that TRP3 channels differ in being store independent. However, condensed cortical F-actin prevented activation of both SOC and TRP3 channels, which suggests that ER-PM interactions underlie coupling of both channels. A cell-permeant inhibitor of inositol trisphosphate receptor (InsP3R) function, 2-aminoethoxydiphenyl borate, prevented both receptor-induced TRP3 activation and store-induced SOC activation. It is concluded that InsP3Rs mediate both SOC and TRP channel opening and that the InsP3R is essential for maintaining coupling between store emptying and physiological activation of SOCs.

Requirement of phospholipase Cdelta4 for the zona pellucida-induced acrosome reaction.

Several phospholipase C (PLC) isoforms have been found in male and female mammalian gametes, and splicing isoforms of PLCdelta4 are predominantly expressed in testis. Here we report that male mice in which the PLCdelta4 gene had been disrupted either produced few small litters or were sterile. In vitro fertilization studies showed that insemination with PLCdelta4-/- sperm resulted in significantly fewer eggs becoming activated and that the calcium transients associated with fertilization were absent or delayed. PLCdelta4-/- sperm were unable to initiate the acrosome reaction, an exocytotic event required for fertilization and induced by interaction with the egg coat, the zona pellucida. These data demonstrate that PLCdelta4 functions in the acrosome reaction that is induced by the zona pellucida during mammalian fertilization.

Expressed cerebellar-type inositol 1,4,5-trisphosphate receptor, P400, has calcium release activity in a fibroblast L cell line.

P400, inositol 1,4,5-trisphosphate receptor (InsP3-R), is a key protein to understanding the mechanisms of inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ mobilization. We obtained the cerebellar-type P400/InsP3-R cDNA and generated an L cell transfectant (L15) that produces cDNA-derived P400/InsP3-R. In membranes, this protein displays high affinity, specificity, and capacity for InsP3, as does the cerebellar P400/InsP3-R. InsP3 can also induce greater 45Ca2+ release from the membrane vesicles of L15 cells than from those of control L cells. These results provide direct evidence that the cDNA-derived P400/InsP3-R protein is actually involved in physiological Ca2+ mobilization, through binding to InsP3 molecules in the same manner as the cerebellar P400/InsP3-R.

Two types of ryanodine receptors in mouse brain: skeletal muscle type exclusively in Purkinje cells and cardiac muscle type in various neurons.

Two types of ryanodine receptors, channels for Ca2+ release from intracellular stores, are known. We detected the skeletal muscle type only in cerebellum by immunoblot analysis of microsomes and partially purified proteins. The cardiac muscle type was found in all parts of the mouse brain. Immunohistochemical study showed that the cardiac muscle type was localized mainly at the somata of most neurons. Analysis of mutant cerebella suggested that the skeletal muscle type was present exclusively in Purkinje cells. These results suggest that Ca(2+)-induced Ca2+ release, probably mediated by the cardiac muscle receptor, functions generally in various neurons, whereas depolarization-induced Ca2+ release, probably mediated by the skeletal muscle receptor, functions specifically in Purkinje cells.

Cloning and expression of a protein kinase C-regulated chloride channel abundantly expressed in rat brain neuronal cells.

cDNA (CIC-3) encoding a protein kinase C-regulated chloride channel was cloned and characterized. The open reading frame encodes 760 amino acids, which possess significantly amino acid identity with previously cloned CIC chloride channels. The chloride currents expressed in Xenopus oocytes injected with CIC-3 cRNA were completely blocked by activation of protein kinase C by 12-O-tetradecanoylphorbol 13-acetate. Abundant expression of CIC-3 mRNA was observed in rat brain, especially in the olfactory bulb, hippocampus, and cerebellum. These findings suggest that CIC-3 may play an important role in neuronal cell function through regulation of membrane excitability by protein kinase C.

Calmodulin mediates calcium-dependent inactivation of the cerebellar type 1 inositol 1,4,5-trisphosphate receptor.

The dependency of purified mouse cerebellar type 1 inositol 1,4,5-trisphosphate receptor (IP3R1)/Ca2+ channel function on cytoplasmic Ca2+ was examined. In contrast to the channels in crude systems, the purified IP3R1 reconstituted into planar lipid bilayers did not show the bell-shaped dependence on Ca2+. It was activated with increasing Ca2+ sublinearly without inhibition even up to 200 microM. The addition of calmodulin to the cytoplasmic side inhibited the channel at high Ca2+ concentrations. Calmodulin antagonists reversed the Ca2+-dependent inactivation of the native channels in cerebellar microsomes. These results indicate that the bell-shaped dependence on cytoplasmic Ca2+ is not an intrinsic property of the IP3R1, and the Ca2+-dependent inactivation is directly mediated by calmodulin.

Glial cell degeneration and hypomyelination caused by overexpression of myelin proteolipid protein gene.

Myelin proteolipid protein (PLP), the major myelin protein in the CNS, has been thought to function in myelin assembly. Thus, mutations within the gene coding for PLP (Plp) cause hypomyelination, such as the jimpy phenotype in mice and Pelizaeus-Merzbacher disease in humans. However, these mutants often exhibit premature death of oligodendrocytes, which form CNS myelin. To elucidate the functional roles of Plp gene products in the maturation and/or survival of oligodendrocytes, we produced transgenic mice overexpressing the Plp gene by introducing extra wild-type mouse Plp genes. Surprisingly, transgenic mice bearing 4 more Plp genes exhibited dysmyelination in the CNS, whereas those with 2 more Plp genes showed normal myelination at an early age (3 weeks after birth), but later developed demyelination. Overexpression of the Plp gene resulted in arrested maturation of oligodendrocytes, and the severity of arrest was dependent on the extent of overexpression. Overexpression also led to oligodendrocyte cell death, apparently caused by abnormal swelling of the Golgi apparatus. Thus, tight regulation of Plp gene expression is necessary for normal oligodendrocyte differentiation and survival, and its overexpression can be the cause of both dys- and demyelination.

The reeler gene-associated antigen on Cajal-Retzius neurons is a crucial molecule for laminar organization of cortical neurons.

In the neurological mutant mouse reeler, the histological organization of the neocortex develops abnormally and essentially results in an inversion of the relative positions of the cortical layers. The reeler mutation, therefore, provides an insight into the molecular mechanisms underlying the formation of the cortical layers. We have generated a monoclonal antibody (CR-50) that probes a distinct allelic antigen present in wild-type but not in reeler mutant mice. CR-50 reacted specifically with Cajal-Retzius neurons, one of the first cortical neurons to differentiate in the neocortex, but whose functional role is not known. When dissociated cerebral cortical cells were incubated with CR-50 in reaggregation culture, the genotype-dependent histogenetic assembly of wild-type cortical cells resembled that of reeler mutants. These findings revealed that the selective expression of a distinct molecule on Cajal-Retzius neurons is critical for the normal lamination of cortical neurons in the mammalian neocortex.

Facilitation of NMDAR-independent LTP and spatial learning in mutant mice lacking ryanodine receptor type 3.

To evaluate the role in synaptic plasticity of ryanodine receptor type 3 (RyR3), which is normally enriched in hippocampal area CA1, we generated RyR3-deficient mice. Mutant mice exhibited facilitated CA1 long-term potentiation (LTP) induced by short tetanus (100 Hz, 100 ms) stimulation. Unlike LTP in wild-type mice, this LTP was not blocked bythe NMDA receptor antagonist D-AP5 but was partially dependent on L-type voltage-dependent Ca2+ channels (VDCCs) and metabotropic glutamate receptors (mGluRs). Long-term depression (LTD) was not induced in RyR3-deficient mice. RyR3-deficient mice also exhibited improved spatial learning on a Morris water maze task. These results suggest that in wild-type mice, in contrast to the excitatory role of Ca2+ influx, RyR3-mediated intracellular Ca2+ ([Ca2+]i) release from endoplasmic reticulum (ER) may inhibit hippocampal LTP and spatial learning.