Osamu Chisaka

Cadherin Regulates Dendritic Spine Morphogenesis

Synaptic remodeling has been postulated as a mechanism underlying synaptic plasticity, and cadherin adhesion molecules are thought to be a regulator of such a process. We examined the effects of cadherin blockage on synaptogenesis in cultured hippocampal neurons. This blockade resulted in alterations of dendritic spine morphology, such as filopodia-like elongation of the spine and bifurcation of its head structure, along with concomitant disruption of the distribution of postsynaptic proteins. The accumulation of synapsin at presynaptic sites and synaptic vesicle recycling were also perturbed, although these synaptic responses to the cadherin blockade became less evident upon the maturation of the synapses. These findings suggest that cadherin regulates dendritic spine morphogenesis and related synaptic functions, presumably cooperating with cadherin-independent adhesive mechanisms to maintain spine-axon contacts.

The Basic Helix-Loop-Helix Factor Olig2 Is Essential for the Development of Motoneuron and Oligodendrocyte Lineages

Sonic hedgehog (Shh), an organizing signal from ventral midline structures, is essential for the induction and maintenance of many ventral cell types in the embryonic neural tube. Olig1 and Olig2 are related basic helix-loop-helix factors induced by Shh in the ventral neural tube. Although expression analyses and gain-of-function experiments suggested that these factors were involved in motoneuron and oligodendrocyte development, they do not clearly define the functional differences between Olig1 and Olig2. We generated mice with a homozygous inactivation of Olig2. These mice did not feed and died on the day of birth. In the spinal cord of the mutant mice, motoneurons are largely eliminated and oligodendrocytes are not produced. Olig2(-/-) neuroepithelial cells in the ventral spinal cord failed to differentiate into motoneurons or oligodendrocytes and expressed an astrocyte marker, S100beta, at the time of oligodendrogenesis. Olig1 or Olig3, other family members, were expressed in the descendent cells that should have expressed Olig2. We concluded that Olig2 is an essential transcriptional regulator in motoneuron and oligodendrocyte development. Our data provide the first evidence that a single gene mutation leads to the loss of two cell types, motoneuron and oligodendrocyte.

Cadherin-11 in Synovial Lining Formation and Pathology in Arthritis

The normal synovium forms a membrane at the edges of joints and provides lubrication and nutrients for the cartilage. In rheumatoid arthritis, the synovium is the site of inflammation, and it participates in an organized tissue response that damages cartilage and bone. We identified cadherin-11 as essential for the development of the synovium. Cadherin-11–deficient mice have a hypoplastic synovial lining, display a disorganized synovial reaction to inflammation, and are resistant to inflammatory arthritis. Cadherin-11 therapeutics prevent and reduce arthritis in mouse models. Thus, synovial cadherin-11 determines the behavior of synovial cells in their proinflammatory and destructive tissue response in inflammatory arthritis.

Functional proteomics identify cornichon proteins as auxiliary subunits of AMPA receptors.

Glutamate receptors of the AMPA-subtype (AMPARs), together with the transmembrane AMPAR regulatory proteins (TARPs), mediate fast excitatory synaptic transmission in the mammalian brain. Here, we show by proteomic analysis that the majority of AMPARs in the rat brain are coassembled with two members of the cornichon family of transmembrane proteins, rather than with the TARPs. Coassembly with cornichon homologs 2 and 3 affects AMPARs in two ways: Cornichons increase surface expression of AMPARs, and they alter channel gating by markedly slowing deactivation and desensitization kinetics. These results demonstrate that cornichons are intrinsic auxiliary subunits of native AMPARs and provide previously unknown molecular determinants for glutamatergic neurotransmission in the central nervous system.

Stability of dendritic spines and synaptic contacts is controlled by alpha N-catenin.

Morphological plasticity of dendritic spines and synapses is thought to be crucial for their physiological functions. Here we show that αN-catenin, a linker between cadherin adhesion receptors and the actin cytoskeleton, is essential for stabilizing dendritic spines in rodent hippocampal neurons in culture. In the absence of αN-catenin, spine heads were abnormally motile, actively protruding filopodia from their synaptic contact sites. Conversely, αN-catenin overexpression in dendrites reduced spine turnover, causing an increase in spine and synapse density. Tetrodotoxin (TTX), a neural activity blocker, suppressed the synaptic accumulation of αN-catenin, whereas bicuculline, a GABA antagonist, promoted it. Furthermore, excess αN-catenin rendered spines resistant to the TTX treatment. These results suggest that αN-catenin is a key regulator for the stability of synaptic contacts.

Loss of Cadherin-11 Adhesion Receptor Enhances Plastic Changes in Hippocampal Synapses and Modifies Behavioral Responses

Cadherins organize symmetrical junctions between the pre- and postsynaptic membranes in central synapses. One of them, cadherin-11 (cad11), is expressed in the limbic system of the brain, most strongly in the hippocampus. Immunohistochemical studies of the hippocampus showed that cad11 proteins were densely distributed in its synaptic neuropil zones; in cultured hippocampal neurons, their distribution often overlapped with that of synaptophysin, and also occasionally with that of GluR1 at spines. To assess the role of cad11 in synaptic formation and/or function, we analyzed brains of cad11-deficient mice. In these mice, long-term potentiation (LTP) in the CA1 region of the hippocampus was, unexpectedly, enhanced; and the level of LTP saturation was increased. In behavioral tests, the mutant mice showed reduced fear- or anxiety-related responses. These results suggest that the cad11-mediated junctions may modulate synaptic efficacy, confining its dynamic changes to a limited range, or these junctions are required for normal development of synaptic organization in the hippocampus.

Targeted disruption of cadherin-11 leads to a reduction in bone density in calvaria and long bone metaphyses

The migration and adhesion of osteoblasts requires several classical cadherins. Cadherin‐11, one of the classical cadherins, was expressed in mouse osteoblasts in skull bone and femur, revealed by immunohistochemistry. To elucidate the function of cadherin‐11 in osteoblastogenesis, cadherin‐11 null mutant mice were investigated. Although apparently normal at birth, Alizarin red staining of null mutant mice showed a reduced calcified area at the frontal suture that caused a round‐shaped calvaria with increasing animal age to 3 months. Consequently, there was a reduction in bone density at the femoral metaphyses and the diploë of calvaria in null mutant mice. In the in vitro culture of newborn calvarial cells, the calcified area of mutant cells was smaller than those derived from wild‐type littermates. These results show that absence of cadherin‐11 leads to reduced bone density in some parts of skeletons including calvaria and long bone metaphyses, and thus suggest that cadherin‐11 plays roles in the regulation of osteoblast differentiation and in the mineralization of the osteoid matrix.

Differential expression of the seven-pass transmembrane cadherin genes Celsr1-3 and distribution of the Celsr2 protein during mouse development

Drosophila Flamingo (Fmi) is an evolutionally conserved seven‐pass transmembrane receptor of the cadherin superfamily. Fmi plays multiple roles in patterning neuronal processes and epithelial planar cell polarity. To explore the in vivo roles of Fmi homologs in mammals, we previously cloned one of the mouse homologs, mouse flamingo1/Celsr2. Here, we report the results of our study of its embryonic and postnatal expression patterns together with those of two other paralogs, Celsr1 and Celsr3. Celsr1–3 expression was initiated broadly in the nervous system at early developmental stages, and each paralog showed characteristic expression patterns in the developing CNS. These genes were also expressed in several other organs, including the cochlea, where hair cells develop planar polarity, the kidney, and the whisker. The Celsr2 protein was distributed at intercellular boundaries in the whisker and on processes of neuronal cells such as hippocampal pyramidal cells, Purkinje cells, and olfactory neurons. Celsr2 is mapped to a distal region of the mouse chromosome 3. We discussed possible functions of seven‐pass transmembrane cadherins in mouse development.

An in vitro system for screening anti-hepatitis b virus drugs

A human hepatoblastoma cell line (HB 611) that continuously synthesizes hepatitis B viral (HBV) DNA was grown in the presence of various inhibitors of DNA synthesis, and the DNA from the cells was analyzed by the Southern blotting method to examine selective inhibition of the viral DNA synthesis. Among those that showed selective inhibition, and interferons alpha and beta, acyclovir, and dideoxy cytidine were effective. This system should be useful for screening new antiviral agents against HBV.

Requirement of the MAP kinase signaling pathways for mouse preimplantation development.

Mammalian preimplantation development involves several crucial events, such as compaction and blastocyst formation, but little is known about essential genes that regulate this developmental process. Here, we have focused on MAP kinase signaling pathways as potential regulatory pathways for the process. Our results show that inhibition of the JNK pathway or of the p38 MAP kinase pathway, but not of the ERK pathway, results in inhibition of cavity formation, and that JNK and p38 are active during mouse preimplantation development. Our subsequent microarray analyses show that, of about 39,000 transcripts analyzed, the number of those genes whose expression level is sensitive to the inhibition of the JNK or the p38 pathway, but insensitive to the inhibition of the ERK pathway, is only 156. Moreover, of the 156 genes, expression of 10 genes (two genes upregulated and eight genes downregulated) is sensitive to either inhibition of the JNK or p38 pathways. These 10 genes include several genes known for their function in axis and pattern formation. Downregulation of some of the 10 genes simultaneously using siRNA leads to abnormality in cavity formation. Thus, this study has successfully narrowed down candidate genes of interest, detailed analysis of which will probably lead to elucidation of the molecular mechanism of preimplantation development.