Biomedical subjects

Suehiro Sakaguchi

Publications and source records attributed to Suehiro Sakaguchi.

12 recordsLinked to original sources

PubMed2006

Immunohistochemical characterization of cell types expressing the cellular prion protein in the small intestine of cattle and mice.

The gastrointestinal tract is thought to be the main site of entry for the pathological isoform of the prion protein (PrP(Sc)). Prion diseases are believed to result from a conformational change of the cellular prion protein (PrP(c)) to PrP(Sc). Therefore, PrP(c) expression is a prerequisite for the infection and spread of the disease to the central nervous system. However, the distribution of PrP(c) in the gut is still a matter of controversy. We therefore investigated the localization of PrP(c) in the bovine and murine small intestine. In cattle, most PrP(c) positive epithelial cells were detected in the duodenum, while a few positive cells were found in the jejunum. PrP(c) was expressed in serotonin producing cells. In bovine Peyer's patches, PrP(c) was distributed in extrafollicular areas, but not in the germinal centre of the jejunum and ileum. PrP(c) was expressed in myeloid lineage cells such as myeloid dendritic cells and macrophages. In mice, PrP(c) was expressed in some epithelial cells throughout the small intestine as well as in cells such as follicular dendritic cell in the germinal centre of Peyer's patches. In this study, we demonstrate that there are a number of differences in the localization of PrP(c) between the murine and bovine small intestines.

Animals↗

PubMed2006

Immunization with recombinant bovine but not mouse prion protein delays the onset of disease in mice inoculated with a mouse-adapted prion.

Host tolerance to endogenous prion protein (PrP) has hampered the development of prion vaccines as PrP is a major component of prions. Indeed, we show that immunization of mice with mouse recombinant PrP elicited no prophylactic effect against a mouse-adapted prion. However, interestingly, mice immunized with recombinant bovine PrP developed the disease significantly later than non-immunized mice after inoculation of a mouse prion. Sheep recombinant PrP exhibited variable prophylactic effects. Mouse recombinant PrP stimulated only very weak antibody responses. In contrast, bovine recombinant PrP was higher immunogenic and produced variable amounts of anti-mouse PrP autoantibodies. Sheep recombinant PrP was also immunogenic but produced more variable amounts of anti-PrP autoantibodies. These results might open a new way for development of prion vaccines.

Animals↗

PubMed2006

Newly established in vitro system with fluorescent proteins shows that abnormal expression of downstream prion protein-like protein in mice is probably due to functional disconnection between splicing and 3' formation of prion protein pre-mRNA.

We and others previously showed that, in some lines of prion protein (PrP)-knockout mice, the downstream PrP-like protein (PrPLP/Dpl) was abnormally expressed in brains partly due to impaired cleavage/polyadenylation of the residual PrP promoter-driven pre-mRNA despite the presence of a poly(A) signal. In this study, we newly established an in vitro transient transfection system in which abnormal expression of PrPLP/Dpl can be visualized by expression of the green fluorescence protein, EGFP, in cultured cells. No EGFP was detected in cells transfected by a vector carrying a PrP genomic fragment including the region targeted in the knockout mice intact upstream of the PrPLP/Dpl gene. In contrast, deletion of the targeted region from the vector caused expression of EGFP. By employing this system with other vectors carrying various deletions or point mutations in the targeted region, we identified that disruption of the splicing elements in the PrP terminal intron caused the expression of EGFP. Recent lines of evidence indicate that terminal intron splicing and cleavage/polyadenylation of pre-mRNA are functionally linked to each other. Taken together, our newly established system shows that the abnormal expression of PrPLP/Dpl in PrP-knockout mice caused by the impaired cleavage/polyadenylation of the PrP promoter-driven pre-mRNA is due to the functional dissociation between the pre-mRNA machineries, in particular those of cleavage/polyadenylation and splicing. Our newly established in vitro system, in which the functional dissociation between the pre-mRNA machineries can be visualized by EGFP green fluorescence, may be useful for studies of the functional connection of pre-mRNA machineries.

3' Untranslated Regions↗

PubMed2006

Enhanced mucosal immunogenicity of prion protein following fusion with B subunit of Escherichia coli heat-labile enterotoxin.

Mucosal vaccine against prion protein (PrP), a major component of prions, is urgently awaited since the oral transmission of prions from cattle to humans is highly suspected. In the present study, we produced recombinant bovine and mouse PrPs fused with or without the B subunit of Escherichia coli heat-labile enterotoxin (LTB) and intranasally immunized mice with these fused proteins. Fusion with LTB markedly enhanced the mucosal immunogenicity of bovine PrP, producing a marked increase in specific IgG and IgA titer in serum. Mouse PrP also showed slightly increased immunogenicity following fusion with LTB. These results demonstrate that LTB-fused PrPs might be potential candidates for protective mucosal prion vaccines.

Administration, Intranasal↗

PubMed2006

Surface plasmon resonance analysis for the screening of anti-prion compounds.

The interaction of anti-prion compounds and amyloid binding dyes with a carboxy-terminal domain of prion protein (PrP121-231) was examined using surface plasmon resonance (SPR) and compared with inhibition activities of abnormal PrP formation in scrapie-infected cells. Most examined compounds had affinities for PrP121-231: antimalarials had low affinities, whereas Congo red, phthalocyanine and thioflavin S had high affinities. The SPR binding response correlated with the inhibition activity of abnormal PrP formation. Several drugs were screened using SPR to verify the findings: propranolol was identified as a new anti-prion compound. This fact indicates that drug screenings by this assay are useful.

Animals↗

PubMed2005

Biological and biochemical characteristics of prion strains conserved in persistently infected cell cultures.

Abnormal prion protein (PrP(Sc)) plays a central role in the transmission of prion diseases, but the molecular basis of prion strains with distinct biological characteristics remains to be elucidated. We analyzed the characteristics of prion disease by using mice inoculated with the Chandler and Fukuoka-1 strains propagated in a cultured mouse neuronal cell line, GT1-7, which is highly permissive to replication of the infectious agents. Strain-specific biological characteristics, including clinical manifestations, incubation period as related to the infectious unit, and pathological profiles, remained unchanged after passages in the cell cultures. We noted some differences in the biochemical aspects of PrP(Sc) between brain tissues and GT1-7 cells which were unlikely to affect the strain phenotypes. On the other hand, the proteinase K-resistant PrP core fragments derived from Fukuoka-1-infected tissues and cells were slightly larger than those from Chandler-infected versions. Moreover, Fukuoka-1 infection, but not Chandler infection, gave an extra fragment with a low molecular weight, approximately 13 kDa, in both brain tissues and GT1-7 cells. This cell culture model persistently infected with different strains will provide a new insight into the understanding of the molecular basis of prion diversity.

Animals↗

PubMed2004

Cellular prion protein regulates intracellular hydrogen peroxide level and prevents copper-induced apoptosis.

The function of cellular prion protein (PrPC), which is a copper binding protein, remains unclear. To elucidate the mechanisms in which PrPC is involved in neuroprotection, we compared death signals in prion protein gene-deficient (Prnp-/-) primary cerebellar granular neurons (CGNs) to those with wild-type (WT) CGNs. When copper was exposed to these CGNs, ZrchI, and Rikn Prnp-/- CGNs were more sensitized and underwent apoptotic cell death more readily than WT CGNs. Furthermore, the level of intracellular hydrogen peroxide (H2O2) in WT CGNs increased by copper toxicity, whereas those in ZrchI and Rikn Prnp-/- CGNs did not. These results suggest that PrPC modulates the intracellular H2O2 level as a copper-binding protein to protect CGNs from apoptotic cell death possibly due to inhibiting a Fenton reaction.

Animals↗

PubMed2004

Doppel-induced Purkinje cell death is stoichiometrically abrogated by prion protein.

Mice devoid of prion protein (PrP) exhibiting ataxia and Purkinje cell degeneration, such as Ngsk Prnp(-/-) mice, ectopically express PrP-like protein, Dpl, in neurons including Purkinje cells. In this study, two types of transgenic (tg) mice expressing Dpl in neurons, tg(N-Dpl), or Purkinje cells only, tg(P-Dpl), were generated on the background of non-ataxic Zrch I Prnp(-/-) mice. In contrast to the tg mice with the Prnp(+/+) background, both tg mice with the Prnp(-/-) alleles developed Purkinje cell degeneration after incubation periods inversely correlated to the levels of Dpl. Some tg mice hemizygous for Prnp allele also developed disease but much later than those carrying the Prnp(-/-) alleles. This indicates that Dpl expressed by Purkinje cells itself is toxic to the cells, and that the neurotoxicity is stoichiometrically antagonized by PrP.

Amyloid↗

PubMed2004

Prion protein suppresses perturbation of cellular copper homeostasis under oxidative conditions.

Prion protein (PrP) binds copper and exhibits superoxide dismutase-like activity, while the roles of PrP in copper homeostasis remain controversial. Using Zeeman graphite furnace atomic absorption spectroscopy, we quantified copper levels in immortalized PrP gene (Prnp)-deficient neuronal cells transfected with Prnp and/or Prnd, which encodes PrP-like protein (PrPLP/Dpl), in the presence or absence of oxidative stress induced by serum deprivation. In the presence of serum, copper levels were not significantly affected by the expression of PrP and/or PrPLP/Dpl, whereas serum deprivation induced a decrease in copper levels that was inhibited by PrP but not by PrPLP/Dpl. The inhibitory effect of PrP on the decrease of copper levels was prevented by overexpression of PrPLP/Dpl. These findings indicate that PrP specifically stabilizes copper homeostasis, which is perturbed under oxidative conditions, while PrPLP/Dpl overexpression prevents PrP function in copper homeostasis, suggesting an interaction of PrP and PrPLP/Dpl and distinct functions between PrP and PrPLP/Dpl on metal homeostasis. Taken together, these results strongly suggest that PrP, in addition to its antioxidant properties, plays a role in stabilizing cellular copper homeostasis under oxidative conditions.

Animals↗

PubMed2003

Cellular prion protein promotes Brucella infection into macrophages.

The products of the Brucella abortus virB gene locus, which are highly similar to conjugative DNA transfer system, enable the bacterium to replicate within macrophage vacuoles. The replicative phagosome is thought to be established by the interaction of a substrate of the VirB complex with macrophages, although the substrate and its host cellular target have not yet been identified. We report here that Hsp60, a member of the GroEL family of chaperonins, of B. abortus is capable of interacting directly or indirectly with cellular prion protein (PrPC) on host cells. Aggregation of PrPC tail-like formation was observed during bacterial swimming internalization into macrophages and PrPC was selectively incorporated into macropinosomes containing B. abortus. Hsp60 reacted strongly with serum from human brucellosis patients and was exposed on the bacterial surface via a VirB complex-associated process. Under in vitro and in vivo conditions, Hsp60 of B. abortus bound to PrPC. Hsp60 of B. abortus, expressed on the surface of Lactococcus lactis, promoted the aggregation of PrPC but not PrPC tail formation on macrophages. The PrPC deficiency prevented swimming internalization and intracellular replication of B. abortus, with the result that phagosomes bearing the bacteria were targeted into the endocytic network. These results indicate that signal transduction induced by the interaction between bacterial Hsp60 and PrPC on macrophages contributes to the establishment of B. abortus infection.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Deletion of N-terminal residues 23-88 from prion protein (PrP) abrogates the potential to rescue PrP-deficient mice from PrP-like protein/doppel-induced Neurodegeneration.

Accumulating evidence has suggested that prion protein (PrP) is neuroprotective and that a PrP-like protein/Doppel (PrPLP/Dpl) is neurotoxic. A line of PrP-deficient mice, Ngsk Prnp0/0, ectopically expressing PrPLP/Dpl in neurons, exhibits late-onset ataxia because of Purkinje cell death that is prevented by a transgene encoding wild-type mouse PrP. To elucidate the mechanisms of neurodegeneration in these mice, we introduced five types of PrP transgene, namely one heterologous hamster, two mouse/hamster chimeric genes, and two mutants, each of which encoded PrP lacking residues 23-88 (MHM2.del23-88) or with E199K substitution (Mo.E199K), into Ngsk Prnp0/0 mice. Only MHM2.del23-88 failed to rescue the mice from the Purkinje cell death. The transgenic mice, MHM2.del23-88/Ngsk Prnp0/0, expressed several times more PrP than did wild-type (Prnp+/+) mice and PrPLP/Dpl at an equivalent level to Ngsk Prnp0/0 mice. Little difference was observed in the pathology and onset of ataxia between Ngsk Prnp0/0 and MHM2.del23-88/Ngsk Prnp0/0. No detergent-insoluble PrPLP/Dpl was detectable in the central nervous system of Ngsk Prnp0/0 mice even after the onset of ataxia. Our findings provide evidence that the N-terminal residues 23-88 of PrP containing the unique octapeptide-repeat region is crucial for preventing Purkinje cell death in Prnp0/0 mice expressing PrPLP/Dpl in the neuron.

Animals↗

PubMed2002

[Prion].

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Animals↗