Tomonari Tsutsumi

Nectin an adhesion molecule involved in formation of synapses

The nectin–afadin system is a novel cell–cell adhesion system that organizes adherens junctions cooperatively with the cadherin–catenin system in epithelial cells. Nectin is an immunoglobulin-like adhesion molecule, and afadin is an actin filament–binding protein that connects nectin to the actin cytoskeleton. Nectin has four isoforms (-1, -2, -3, and -4). Each nectin forms a homo-cis-dimer followed by formation of a homo-trans-dimer, but nectin-3 furthermore forms a hetero-trans-dimer with nectin-1 or -2, and the formation of each hetero-trans-dimer is stronger than that of each homo-trans-dimer. We show here that at the synapses between the mossy fiber terminals and dendrites of pyramidal cells in the CA3 area of adult mouse hippocampus, the nectin–afadin system colocalizes with the cadherin–catenin system, and nectin-1 and -3 asymmetrically localize at the pre- and postsynaptic sides of puncta adherentia junctions, respectively. During development, nectin-1 and -3 asymmetrically localize not only at puncta adherentia junctions but also at synaptic junctions. Inhibition of the nectin-based adhesion by an inhibitor of nectin-1 in cultured rat hippocampal neurons results in a decrease in synapse size and a concomitant increase in synapse number. These results indicate an important role of the nectin–afadin system in the formation of synapses.

Neural circuits and functional organization of the striatum

Abstract The basal ganglia and motor thalamic nuclei are functionally and anatomically divided into the sensorimotor, supplementary motor, premotor, associative and limbic territories. There exist both primary segregated basal ganglia-thalamocortical loops and convergence of functionally related information from different cortical areas onto these cortical basal gaglia-thalamocortical loops. The basal ganglia-thalamocortical loop arising from the sensorimotor area, supplementary motor area (SMA), premotor area and cingulate motor area provides distinct segregated subloops through the functionally distict stritial, pallidal and thalamic regions with partial overlap. The subthalamic nucleus (STN) is also topographically organized. The ventrolateral part of the caudal 2/3 levels of the medial pallidal segment (GPi) projects to the primary motor area via the oral part of the ventral lateral thalamic nucleus (VLo) (Voa, Vop by Hasslers nomenclature). The thalamic relay nuclei of the GPi projection to SMA are identified in the transitional zoe of the VApc (parvicellular part of the anterior ventral nucleus)-VLo and in the rostromedical part of the VLo. The thalamic nuclei relaying the cingulate subloop are not yet clearly defined. The supplementary motor subloop appears to be divided into the pre-SMA and SMA proper subloops. The premotor area is also divided into the dorsal premotor area subloop and the ventral premotor area subloop. It is suggested that the limbic loop consists of a number of subloops in the monkey as indicated by Haber et al. [67] and in rats [64]. We review here the microcircuitry of the striatum, as well as the convergence and integration between the functionally segregated loops. Finally, we discuss the functional implications of stritial connections.

Synaptic Scaffolding Molecule α Is a Scaffold To Mediate N-Methyl-d-Aspartate Receptor-Dependent RhoA Activation in Dendrites

ABSTRACT Synaptic scaffolding molecule (S-SCAM) interacts with a wide variety of molecules at excitatory and inhibitory synapses. It comprises three alternative splicing variants, S-SCAMα, -β, and -γ. We generated mutant mice lacking specifically S-SCAMα. S-SCAMα-deficient mice breathe and feed normally but die within 24 h after birth. Primary cultured hippocampal neurons from mutant mice have abnormally elongated dendritic spines. Exogenously expressed S-SCAMα corrects this abnormal morphology, while S-SCAMβ and -γ have no effect. Active RhoA decreases in cortical neurons from mutant mice. Constitutively active RhoA and ROCKII shift the length of dendritic spines toward the normal level, whereas ROCK inhibitor (Y27632) blocks the effect by S-SCAMα. S-SCAMα fails to correct the abnormal spine morphology under the treatment of N-methyl-d-aspartate (NMDA) receptor inhibitor (AP-5), Ca2+/calmodulin kinase inhibitor (KN-62), or tyrosine kinase inhibitor (PP2). NMDA treatment increases active RhoA in dendrites in wild-type hippocampal neurons, but not in mutant neurons. The ectopic expression of S-SCAMα, but not -β, recovers the NMDA-responsive accumulation of active RhoA in dendrites. Phosphorylation of extracellular signal-regulated kinase 1/2 and Akt and calcium influx in response to NMDA are not impaired in mutant neurons. These data indicate that S-SCAMα is a scaffold required to activate RhoA protein in response to NMDA receptor signaling in dendrites.

A Novel Mode of Action of an ArfGAP, AMAP2/PAG3/Papα, in Arf6 Function

Previously we reported that AMAP2/PAG3/Papα/KIAA0400, a GTPase-activating protein (GAP), acts to antagonize Arf6 function when overexpressed, whereas it was shown to exhibit efficient GAP activities for other Arf isoforms in vitro. Here, we found that AMAP2, through its ArfGAP domain, binds to GTP-Arf6 but not to GDP-Arf6 or other Arfs irrespective of nucleotide status. The majority of AMAP2 was localized to intracellular tubulovesicular structures and redistributed to Arf6-enriched membrane areas upon Arf6 activation. In HeLa cells, Arf6 has been shown to be involved in the clathrin-independent endocytosis of Tac, but not the clathrin-dependent endocytosis of transferrin. We found that Arf6 silencing inhibited the internalization of Tac, but not transferrin, in HeLa cells. Internalization of Tac, but not transferrin, was also significantly inhibited by AMAP2 silencing and overexpression. AMAP2 was moreover found to bind to amphiphysin IIm, a component of the endocytic machinery, via its proline-rich domain. We propose that AMAP2 has dual mechanisms for its function; it exhibits efficient catalytic GAP activity for the class I and II Arfs and yet is involved in the cellular function of the class III Arf without immediate GAP activity. These dual mechanisms of AMAP2 may be important for the cellular function of GTP-Arf6.

PASK (proline-alanine-rich Ste20-related kinase) binds to tubulin and microtubules and is involved in microtubule stabilization.

Proline-alanine-rich Ste20-related kinase (PASK, also referred to as SPAK) has been linked to ion transport regulation. Here, we report two novel activities of PASK: binding to tubulin and microtubules and the promotion of microtubule assembly. Tubulin binding assay showed that full-length PASK and its kinase domain bound to purified tubulin whereas the N-terminal or C-terminal non-catalytic domains of PASK did not. The full-length PASK and its kinase domain were sedimented with paclitaxel-stabilized microtubules by ultracentrifugation. These results indicate that the kinase domain of PASK can interact directly with both microtubules and soluble tubulin in vitro. Truncated PASK lacking the N-terminal non-catalytic domain promoted microtubule assembly at a subcritical concentration of purified tubulin. FLAG-PASK expressed in COS-7 cells translocated to the cytoskeleton when the cells were stimulated with hypertonic sodium chloride, and stabilized microtubules against depolymerization by nocodazole. Our findings suggest that PASK may regulate the cytoskeleton by modulating microtubule stability.

Distribution of Pontomesencephalic Neurons Projecting to the Medullary Reticular Areas and Spinal Cord in Relation to the Pedunculopontine Nucleus in the Monkey

The pedunculopontine tegmental nucleus (PPN) is located in the mesopontine tegmentum lateral to the decussation of the superior cerebellar peduncle, at the levels from the caudal pole of the red nucleus to the ponto-mesencephalic junction, and innervated by descending projections from the motor outflow of the basal ganglia. It is interconnected with the substantia nigra (SN), the internal segment of pallidum (GPi) and the subthalamic nucleus (STN), and is an important interface between the basal ganglia and the brainstem motor system (Jackson and Crossman, 1983; Garcia-Rilli et al., 1991; Rye et al., 1996; Winn et al., 1997). It also receives cortical afferents mainly from the primary motor cortex, and afferents from the nucleus accumbens directly or indirectly via its output stations, such as the ventral pallidum, lateral hypothalamic area, and the ventral tegmental area (VTA) (Garcia-Rill et al., 1983c). The PPN transfers limbic information concerned with motivation reinforcement from the ventral striatum to the basal ganglia thalamo-cortical loops and to the ponto-medullary systems (limbic-motor integration). The PPN could be involved in locomotion and a variety of behavioral functions, such as the control of sleep and wake, learning and reinforcement processes, and autonomic functions. It is also involved in response choice to interrupt ongoing behavior; either to switch to a new response or simply to cease the current response (Winn et al., 1997).

Characterization of Ste20-related protein kinase PASK

The major PKG substrates found in the vascular smooth muscle, GO (2 15 250-kDa) and GI (120 I40-kDa), were compared with type I IP3 receptor (IP3R, 240-kDa) and myosin-binding subunit (MBS, 138-kDa) of myosin light chain phosphatase, PKG substrates which we identified in vascular smooth muscle as those implicated in the cGMP-induced relaxation of vascular smooth muscle. To identify molecular nature of GO and GI, microsomal GO and GI were thiophosphorylated with [y-“S]ATP, and then subjected to immunoprecipitation with affinity-purified antibodies (Abs) specific to IP3R and MBS. Immunoprecipitation with an anti-MBS Ab resulted in removal of a minor portion of GI, while an antiIP3R Ab precipitated GO completely. Unexpectedly, the anti-IP3R Ab co-precipitated a large part of Gl, which could not be recognized on Western blotting by anti-MB& anti-IP3R or anti-plasma membrane Ca’*-pump Abs. Furthermore, just like IP3R, GO was found to bind calmodulin-column in a Ca*‘-dependent manner. GI was also co-purified with GO in the column chromatography. These results suggested that GO is identical to type 1 IP3R, and that GI consists of several polypeptides in which MBS represents a minor component, and the protein associated with IP3R is a major component.

616 Localization and characterization of a novel Ste20-related protein kinase

Tomonari Tsutsumi, Hiroshi Ushiro, Tetsuro Kayahara, Katsuma Nakano We have cloned a novel protein kinase cDNA from rat brain. The deduced amino acid sequence of the catalytic domain indicates that it belongs to the Ste20-related protein kinase family, the members of which have been reported to act upstream of MAP kinase pathways. The distribution of the kinase in rat organs was examined with a rabbit polyclonal antibody against the C-terminal non-catalytic region. Immunoblot analysis demonstrated ubiquitous expression of the enzyme, except in liver and skeletal muscle where no immunoreactive band was detected. Immunohistochemical analysis revealed cell type specific distribution of the enzyme. In the brain, the most prominent immunoreactivity was found in epithelial cells of choroid plexus. A moderate immunoreactivity was seen in large neurons such as motor neurons in brainstem, cerebellar Purkinje cells and pyramidal cells of cerebral cortex.

704 Molecular cloning of a novel protein kinase cDNA from rat brain

Merkel cells are mechano-receptor cells in the skin that form synaptic contact with primary sensory nerve fibers. Culturing of these cells in vitro has not been successful. We report here that an almost pure culture of Merkel cells in chemically-defined media was achieved, and that these cells underwent apoptosis during culture in these media. In order to obtained Merkel cells, we dissected cores of hair follicles located in the upper-lips of the rat under binocular microscope, and then, they were digested with collagenase in L-15 medium. Merkel cells were isolated from the cores by pippeting, and were platted on poly-L -lysine-coated plastic dishes. Merkel cells were identified by staining with a fluorescence marking dye, quinacrine. During culture in the medium, population of the Merkel cells reduced quickly in time. Addition of serum (5 %) or actinomycin D (2 nM) in the medium prevented the reduction in the cell population. Furthermore, nuclei of many Merkel cells were well stained with TUNEL-method. We thus concluded that the loss of Merkel cells during culture in the serum-free medium was due to the programmed cell death.

1612 Cerebello-thalamic projections in monkeys with special reference to the rostral regions of motor thalamic nuclei

A number of questions still persist with regard to the cerebellar projections to the rostra1 regions of motor thalamic nuclei. Attempts were made to confirm the existence of these projections, and to determine their cells of origin in the cerebellar nuclei in macaque monkeys using axonai transport techniques. Biotinylated dextran amine (BDA) was injected into the various parts of distinct cerebellar nuclei in one group, and wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) injected in the subdivisions of motor thalamic nuclei in the other group. Our data indicates following major findings: The nucleus ventralis lateralis pars oralis (VLo) received cerebellar afferents mainly from the ventromedial portion of nucleus dentatus (NL). These cerebellar projections terminated in the lateral region of VLo. The nucleus ventralis posterolatemlis pars oralis (VPLo) received afferents from all crebellar nuclei. The major VPLo input arose from the nucleus interpositus anterior (NIA) and the dorsal to medial parts of the nucleus interpositus posterior (NIP). A second heavy input arose from the dorsal half of rostra1 NL, and sparser input was present from the nucleus fastigii (NM). Area X received cerebellar afferents from the NIP, NM and whole parts of the caudal NL as well as the ventral intermediate part of middle NL. Cerebellar projections to the nucleus lateralis posterior (LP) were also traced from NIA. No terminal labeling was seen in the centromedian nucleus.