Ryuji Nomura

Human Coronavirus 229E Binds to CD13 in Rafts and Enters the Cell through Caveolae

ABSTRACT CD13, a receptor for human coronavirus 229E (HCoV-229E), was identified as a major component of the Triton X-100-resistant membrane microdomain in human fibroblasts. The incubation of living fibroblasts with an anti-CD13 antibody on ice gave punctate labeling that was evenly distributed on the cell surface, but raising the temperature to 37°C before fixation caused aggregation of the labeling. The aggregated labeling of CD13 colocalized with caveolin-1 in most cells. The HCoV-229E virus particle showed a binding and redistribution pattern that was similar to that caused by the anti-CD13 antibody: the virus bound to the cell evenly when incubated on ice but became colocalized with caveolin-1 at 37°C; importantly, the virus also caused sequestration of CD13 to the caveolin-1-positive area. Electron microscopy confirmed that HCoV-229E was localized near or at the orifice of caveolae after incubation at 37°C. The depletion of plasmalemmal cholesterol with methyl β-cyclodextrin significantly reduced the HCoV-229E redistribution and subsequent infection. A caveolin-1 knockdown by RNA interference also reduced the HCoV-229E infection considerably. The results indicate that HCoV-229E first binds to CD13 in the Triton X-100-resistant microdomain, then clusters CD13 by cross-linking, and thereby reaches the caveolar region before entering cells.

Neurotropism of swine haemagglutinating encephalomyelitis virus (coronavirus) in mice depending upon host age and route of infection.

Abstract Mice aged 1, 4 or 8 weeks were inoculated with haemagglutinating encephalomyelitis virus (HEV), strain 67N, by the intracerebral (i.c.), intranasal (i.n.), intraperitoneal (i.p.), subcutaneous (s.c.), intravenous (i.v.) or oral route, with different doses. In 1-week-old mice, mortality and mean time to death were mostly the same regardless of the inoculation route, except for the oral route, which appeared to be the least effective. The virus killed 4-week-old mice readily by all routes of inoculation except the oral, and 8-week-old mice by i.c., i.n. or s.c. inoculation. In descending order of efficacy, the routes of HEV infection were: i.c., i.n., s.c., i.p., i.v. and oral. To follow the spread of HEV from peripheral nerves to the central nervous system (CNS), the virus was inoculated subcutaneously into the right hind leg of 4-week-old mice. The virus was first detected in the spinal cord on day 2, and in the brain on day 3. The brain titres became higher than those of the spinal cord, reaching a maximum of 107PFU/0.2 g when the animals were showing CNS signs. Viral antigen was first detected immunohistochemically in the lumbar spinal cord and the dorsal root ganglion ipsilateral to the inoculated leg; it was detected later in the pyramidal cells of the hippocampus and cerebral cortex, and in the Purkinje cells of the cerebellum but not in the ependymal cells, choroid plexus cells or other glial cells. The infected neurons showed no cytopathological changes.

Targeted deletion of the C-terminus of the mouse adenomatous polyposis coli tumor suppressor results in neurologic phenotypes related to schizophrenia.

BackgroundLoss of adenomatous polyposis coli (APC) gene function results in constitutive activation of the canonical Wnt pathway and represents the main initiating and rate-limiting event in colorectal tumorigenesis. APC is likely to participate in a wide spectrum of biological functions via its different functional domains and is abundantly expressed in the brain as well as in peripheral tissues. However, the neuronal function of APC is poorly understood. To investigate the functional role of Apc in the central nervous system, we analyzed the neurological phenotypes of Apc1638T/1638T mice, which carry a targeted deletion of the 3′ terminal third of Apc that does not affect Wnt signaling.ResultsA series of behavioral tests revealed a working memory deficit, increased locomotor activity, reduced anxiety-related behavior, and mildly decreased social interaction in Apc1638T/1638T mice. Apc1638T/1638T mice showed abnormal morphology of the dendritic spines and impaired long-term potentiation of synaptic transmission in the hippocampal CA1 region. Moreover, Apc1638T/1638T mice showed abnormal dopamine and serotonin distribution in the brain. Some of these behavioral and neuronal phenotypes are related to symptoms and endophenotypes of schizophrenia.ConclusionsOur results demonstrate that the C-terminus of the Apc tumor suppressor plays a critical role in cognitive and neuropsychiatric functioning. This finding suggests a potential functional link between the C-terminus of APC and pathologies of the central nervous system.

Requirement of the tumour suppressor APC for the clustering of PSD‐95 and AMPA receptors in hippocampal neurons

Mutations in the adenomatous polyposis coli (APC) gene are associated with familial adenomatous polyposis and sporadic colorectal tumours. The APC gene is expressed ubiquitously in various tissues, especially throughout the large intestine and central nervous system (CNS). In the CNS, the expression of the APC protein is highest during embryonic and early postnatal development. APC associates through its C‐terminal region with postsynaptic density (PSD)‐95, a neuronal protein that participates in synapse development. Here, we examined the involvement of APC in synaptogenesis. In cultured hippocampal neurons, both overexpression of a dominant‐negative construct that disrupts the APC–PSD‐95 interaction and knockdown of APC expression using small interfering RNA (siRNA) inhibited the clustering of PSD‐95 and a glutamate receptor subunit, and reduced alpha‐amino‐3‐hydroxy‐5‐methyl‐isoxazole‐4‐propionate (AMPA)‐induced activity of AMPA receptors; however, the clustering of an N‐methyl‐d‐aspartate (NMDA) receptor subunit was unaffected. These results are suggestive of APC involvement in the development of glutamatergic synapses.

Neuronal Spread of Swine Hemagglutinating Encephalomyelitis Virus (HEV) 67N Strain in 4-Week-Old Rats

The HEV 67N strain causes encephalomyelitis or vomiting and wasting syndrome in piglets 1,2. In experimental infection of piglets, the virus spreads along the nerve pathways to the central nervous system (CNS), and is restricted to the neurons. In our experimental studies of HEV 67N strain, the virus produced encephalitis in mice when inoculated by several routes, and propagated mainly in the neurons in the CNS 4. However, 20-day-old or older mice were resistant to the virus inoculated by intravenous (i.v.), intraperitoneal (i.p.) or subcutaneous (s.c.) route. In contrast, 4-week-old rats died of encephalitis after i.v., i.p. and s.c. as well as intracerebral (i.c.) and intranasal (i.n.) inoculation. However, when rats were inoculated by s.c. route, they died a few days earlier than those by i.p. and i.v. routes, suggesting that the virus might be spread to the CNS by neural routes rather than blood stream. To see the virus spread from the peripheral nerve to the brain, the virus was directly inoculated in to the sciatic nerve of rats. These studies demonstrated that HEV spread from the sciatic nerve to the brain by the neural route, and that persistent infection of HEV was established in rats6.

Requirement of DLG1 for cardiovascular development and tissue elongation during cochlear, enteric, and skeletal development: Possible role in convergent extension

The Dlg1 gene encodes a member of the MAGUK protein family involved in the polarization of epithelial cells. Null mutant mice for the Dlg1 gene (Dlg1-/- mice) exhibit respiratory failure and cyanosis, and die soon after birth. However, the cause of this neonatal lethality has not been determined. In the present study, we further examined Dlg1-/- mice and found severe defects in the cardiovascular system, including ventricular septal defect, persistent truncus arteriosus, and double outlet right ventricle, which would cause the neonatal lethality. These cardiovascular phenotypes resemble those of mutant mice lacking planar cell polarity (PCP) genes and support a recent notion that DLG1 is involved in the PCP pathway. We assessed the degree of involvement of DLG1 in the development of other organs, as the cochlea, intestine, and skeleton, in which PCP signaling has been suggested to play a role. In the organ of Corti, tissue elongation was inhibited accompanied by disorganized arrangement of the hair cell rows, while the orientation of the stereocilia bundle was normal. In the sternum, cleft sternum, abnormal calcification pattern of cartilage, and disorganization of chondrocytes were observed. Furthermore, shortening of the intestine, sternum, and long bones of the limbs was observed. These phenotypes of Dlg1-/- mice involving cellular disorganization and insufficient tissue elongation strongly suggest a defect in the convergent extension movements in these mice. Thus, our present results provide a possibility that DLG1 is particularly required for convergent extension among PCP signaling-dependent processes.

The C-terminal domain of the adenomatous polyposis coli (Apc) protein is involved in thyroid morphogenesis and function

Adenomatous polyposis coli (APC) is a multifunctional protein as well as a tumor suppressor. To determine the functions of the C-terminal domain of Apc, we have investigated Apc1638T/1638T mice, which express a truncated Apc that lacks the C-terminal domain. Apc1638T/1638T mice are tumor free and exhibit growth retardation. In the present study, we analyzed the morphology and functions of the thyroid gland in Apc1638T/1638T mice. There was no significant difference in the basal concentration of serum thyroid hormones between Apc1638T/1638T and Apc+/+ mice. Thyroid follicle size was significantly larger in Apc1638T/1638T mice than in Apc+/+ mice. The extent of serum T4 elevation following exogenous thyroid-stimulating hormone (TSH) injection was lower in Apc1638T/1638T mice than in Apc+/+ mice. TSH also induced a greater reduction in thyroid follicle size in Apc1638T/1638T mice than in Apc+/+ mice. Analyses using immunohistochemistry and electron microscopy indicated that follicular epithelial cells in Apc1638T/1638T mice had an enlarged rough endoplasmic reticulum of irregular shape. These results suggest that the C-terminal domain of Apc is involved in thyroid morphology and function.

Identification of DNA-dependent protein kinase catalytic subunit as a novel interaction partner of lymphocyte enhancer factor 1

Lymphocyte enhancer factor 1 (LEF1), a member of the LEF/T-cell-specific factor (TCF) family of the high mobility group domain transcription factors, acts downstream in canonical Wnt signaling. Aberrant transactivation of LEF1 contributes to the tumorigenesis of colonic neoplasms, sebaceous skin tumors, and lymphoblastic leukemia. LEF1-associated proteins are crucial for regulating its transcriptional activity. In this study, glutathione-S-transferase pull-down assay and mass spectrometry enabled identification of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a novel interaction partner for LEF1. The interaction between LEF1 and DNA-PKcs was confirmed using in vivo co-immunoprecipitation. Furthermore, double immunofluorescence observations showed that LEF1 and DNA-PKcs colocalized in the nuclei of colon adenocarcinoma cell lines. Identification of the interaction between LEF1 and DNA-PKcs may provide clues for a novel therapy for cancer treatment as well as for understanding LEF1-mediated transcriptional regulation.

Some fine-structural findings on the thyroid gland in Apc1638T/1638T mice that express a C-terminus lacking truncated Apc

Adenomatous polyposis coli (Apc) is a multifunctional protein as well as a tumor suppressor. To determine the functions of the C-terminal domain of Apc, we examined Apc1638T/1638T mice that express a truncated Apc lacking the C-terminal domain. The Apc1638T/1638T mice were tumor free and exhibited growth retardation. We recently reported abnormalities in thyroid morphology and functions of Apc1638T/1638T mice, although the mechanisms underlying these abnormalities are not known. In the present study, we further compared thyroid gland morphology in Apc1638T/1638T and Apc+/+ mice. The diameters of thyroid follicles in the left and right lobes of the same thyroid gland of Apc1638T/1638T mice were significantly different whereas the Apc+/+ mice showed no significant differences in thyroid follicle diameter between these lobes. To assess the secretory activities of thyroid follicular cells, we performed double-immunostaining of thyroglobulin, a major secretory protein of these cells, and the rough endoplasmic reticulum (rER) marker calreticulin. In the Apc1638T/1638T follicular epithelial cells, thyroglobulin was mostly colocalized with calreticulin whereas in the Apc+/+ follicular epithelial cells, a significant amount of the cytoplasmic thyroglobulin did not colocalize with calreticulin. In addition, in thyroid-stimulating hormone (TSH)-treated Apc1638T/1638T mice, electron microscopic analysis indicated less frequent pseudopod formation at the apical surface of the thyroid follicular cells than in Apc+/+ mice, indicating that reuptake of colloid droplets containing iodized thyroglobulin is less active. These results imply defects in intracellular thyroglobulin transport and in pseudopod formation in the follicular epithelial cells of Apc1638T/1638T mice and suggest suppressed secretory activities of these cells.

γ-Tubulin-like molecules in the mouse duodenal epithelium

A mouse monoclonal antibody (G9, Horio et al. in Cell Motil Cytoskel 44:284–295, 1999) that was raised against the γ-tubulin from a fission yeast, Schizosaccharomyces pombe, showed a unique staining in the mouse small intestine. Similar to another anti-γ-tubulin antibody that is commercially available, G9 showed typical dot-like staining corresponding to the microtubule-organizing center in the free cells of the epithelium and the connective tissue under it. In addition, G9 stained the cell–cell contacts in the epithelium. This stained region was not bicellular but tricellular junctions of the enterocytes. This staining was unique to G9 and was diminished on the sample of the mouse small intestine, which had lost most of its filamentous microtubules through the preparation process. The tricellular junction is thought to be the weakest point of the epithelial barrier, and no other junctional structures have been identified except for the central sealing elements extending from the tight junctions between the two cells. Our results suggest the existence of a new molecule underlying the tricellular junctions, which may relate to γ-tubulin and the microtubules.