Suehiro Sakaguchi

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.

Physiological Expression of the Gene for PrP-Like Protein, PrPLP/Dpl, by Brain Endothelial Cells and its Ectopic Expression in Neurons of PrP-Deficient Mice Ataxic Due to Purkinje Cell Degeneration

Recently, a novel gene encoding a prion protein (PrP)-like glycoprotein, PrPLP/Dpl, was identified as being expressed ectopically by neurons of the ataxic PrP-deficient (PRNP(-/-)) mouse lines exhibiting Purkinje cell degeneration. In adult wild-type mice, PrPLP/Dpl mRNA was physiologically expressed at a high level by testis and heart, but was barely detectable in brain. However, transient expression of PrPLP/Dpl mRNA was detectable by Northern blotting in the brain of neonatal wild-type mice, showing maximal expression around 1 week after birth. In situ hybridization paired with immunohistochemistry using anti-factor VIII serum identified brain endothelial cells as expressing the transcripts. Moreover, in the neonatal wild-type mice, the PrPLP/Dpl mRNA colocalized with factor VIII immunoreactivities in spleen and was detectable on capillaries in lamina propria mucosa of gut. These findings suggested a role of PrPLP/Dpl in angiogenesis, in particular blood-brain barrier maturation in the central nervous system. Even in the ataxic Ngsk PRNP(-/-) mice, the physiological regulation of PrPLP/Dpl mRNA expression in brain endothelial cells was still preserved. This strongly supports the argument that the ectopic expression of PrPLP/Dpl in neurons, but not deregulation of its physiological expression in endothelial cells, is involved in the neuronal degeneration in ataxic PRNP(-/-) mice.

Identification of a novel gene encoding a PrP-like protein expressed as chimeric transcripts fused to PrP exon 1/2 in ataxic mouse line with a disrupted PrP gene.

Abstract1. Mouse lines lacking prion protein (PrPC) have a puzzling phenotypic discrepancy. Some, but not all, developed late-onset ataxia due to Purkinje cell degeneration.2. Here, we identified aberrant mRNA species in the brain of Ngsk Prnp0/0 ataxic, but not in nonataxic Zrch Prnp0/0 mouse line. These mRNAs were chimeric between the noncoding exons 1 and 2 of the PrP gene (Prnp) and the novel sequence encoding PrP-like protein (PrPLP), a putative membrane glycoprotein with 23% identity to PrPC in the primary amino acid structure. The chimeric mRNAs were generated from the disrupted Prnp locus of Ngsk Prnp0/0 mice lacking a part of the Prnp intron 2 and its splice acceptor signal.3. In the brain of wild-type and Zrch Prnp0/0 mice, PrPLP mRNA was barely detectable. In contrast, in the brain of Ngsk Prnp0/0 mice, PrP/PrPLP chimeric mRNAs were expressed in neurons, at a particularly high level in hippocampus pyramidal cells and Purkinje cells under the control of the Prnp promoter.4. In addition to the functional loss of PrPC, ectopic PrPLP expression from the chimeric mRNAs could also be involved in the Purkinje cell degeneration in Ngsk Prnp0/0 mice.

Upregulation of the Genes Encoding Lysosomal Hydrolases, a Perforin-Like Protein, and Peroxidases in the Brains of Mice Affected with an Experimental Prion Disease

ABSTRACT In an attempt to identify the molecules involved in the pathogenesis of prion diseases, we performed cDNA subtraction on the brain tissues of mice affected with an experimental prion disease and the unaffected control. The genes identified as being upregulated in the prion-affected brain tissue included those encoding a series of lysosomal hydrolases (lysozyme M and both isoforms of β-N-acetylhexosaminidase), a perforin-like protein (macrophage proliferation-specific gene-1 [MPS-1]), and an oxygen radical scavenger (peroxiredoxin). Dramatic increases in the expression level occurred at between 12 and 16 weeks after intracerebral inoculation of the prion, coinciding with the onset of spongiform degeneration. The proteinase K-resistant prion protein (PrPSc) became detectable by immunoblotting well before 12 weeks, suggesting a causal relationship between this and the gene activation. Immunohistochemistry paired with in situ hybridization on sections of the affected brain tissue revealed that expression of the peroxiredoxin gene was detectable only in astrocytes and was noted throughout the affected brain tissue. On the other hand, the genes for the lysosomal hydrolases and MPS-1 were overexpressed exclusively by microglia, which colocalized with the spongiform morphological changes. A crucial role for microglia in the spongiform degeneration by their production of neurotoxic substances, and possibly via the aberrant activation of the lysosomal system, would have to be considered.

Kinetics of infectivity are dissociated from PrP accumulation in salivary glands of Creutzfeldt-Jakob disease agent-inoculated mice

The protease-resistant isoform of prion protein (PrP) has been implicated in the pathogenesis and transmission of Creutzfeldt-Jakob disease (CJD), scrapie and other related diseases, but the relationship between the infectious agent and PrP awaits elucidation. In the present study, we have examined levels of infectivity together with accumulation of the protease-resistant form of PrP (PrPCJD) in various tissues of CJD agent-inoculated mice. Accumulation of PrPCJD occurred only in tissues, including brain, salivary gland and spleen, in which infectivity was readily detectable throughout the course of the experiment. The brain showed the highest levels of both infectivity and PrPCJD accumulation, with well correlated kinetics. On the other hand, the high titres of infectivity detected in salivary gland and spleen early after inoculation of the agent were obviously distinguishable from PrPCJD. Furthermore, in the salivary gland, the kinetics of infectivity and the accumulation of PrPCJD reversed; infectivity declined as PrPCJD accumulated in the tissue. Our findings indicate that PrPCJD accumulation is associated with replication of the agent; however, PrPCJD is unlikely to be the agent itself.

PrP fragment 106-126 is toxic to cerebral endothelial cells expressing PrP(C).

A hydrophobic, fibrillogenic peptide fragment of human prion protein (PrP 106–126) had in vitro toxicity to neurons expressing cellular prion protein (PrPC). In this study, we proved that primary cultures of mouse cerebral endothelial cells (MCEC) express PrPC. Incubation of MCEC with PrP 106–126 (25–200 μM) caused a dose-dependent toxicity assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase release, bis-benzimide staining for nuclear morphology, and trypan blue exclusion test. Pentosan polysulphate (50–100 μg/ml), a drug effective in scrapie prophylaxis, dose-dependently attenuated the injury. MCEC cultures from mice homogenous for the disrupted PrP gene were resistant to the toxicity of PrP 106–126. In conclusion, cerebral endothelium expressing PrPC may be directly damaged during spongiform encephalopathies.

Prions disturb post-Golgi trafficking of membrane proteins

Conformational conversion of normal cellular prion protein PrP(C) into pathogenic PrP(Sc) is central to the pathogenesis of prion diseases. However, the pathogenic mechanism remains unknown. Here we show that post-Golgi vesicular trafficking is significantly delayed in prion-infected N2a cells. Accordingly, cell surface expression of membrane proteins examined, including PrP(C), insulin receptor involved in neuroprotection, and attractin, whose mutation causes prion disease-like spongiform neurodegeneration, is reduced. Instead, they accumulate in the Golgi apparatus. PrP(Sc) is detected throughout endosomal compartments, being particularly abundant in recycling endosome. We also show reduced surface expression of PrP(C) and insulin receptor in prion-infected mouse brains well before the onset of disease. These results suggest that prion infection might impair post-Golgi trafficking of membrane proteins to the cell surface in neurons via PrP(Sc) accumulated in recycling endosome, and eventually induce neuronal dysfunctions associated with prion diseases.

Orally Administered Prion Protein Is Incorporated by M Cells and Spreads into Lymphoid Tissues with Macrophages in Prion Protein Knockout Mice

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases. Infection by the oral route is assumed to be important, although its pathogenesis is not understood. Using prion protein (PrP) knockout mice, we investigated the sequence of events during the invasion of orally administered PrPs through the intestinal mucosa and the spread into lymphoid tissues and the peripheral nervous system. Orally administered PrPs were incorporated by intestinal epitheliocytes in the follicle-associated epithelium and villi within 1 hour. PrP-positive cells accumulated in the subfollicle region of Peyers patches a few hours thereafter. PrP-positive cells spread toward the mesenteric lymph nodes and spleen after the accumulation of PrPs in the Peyers patches. The number of PrP molecules in the mesenteric lymph nodes and spleen peaked at 2 days and 6 days after inoculation, respectively. The epitheliocytes in the follicle-associated epithelium incorporating PrPs were annexin V-positive microfold cells and PrP-positive cells in Peyers patches and spleen were CD11b-positive and CD14-positive macrophages. Additionally, PrP-positive cells in Peyers patches and spleen were detected in the vicinity of peripheral nerve fibers in the early stages of infection. These results indicate that orally delivered PrPs were incorporated by microfold cells promptly after challenge and that macrophages might act as a transporter of incorporated PrPs from the Peyers patches to other lymphoid tissues and the peripheral nervous system.

Biological and Biochemical Characteristics of Prion Strains Conserved in Persistently Infected Cell Cultures

ABSTRACT Abnormal prion protein (PrPSc) 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 PrPSc 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, ∼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.

Female-specific neuroprotection against transient brain ischemia observed in mice devoid of prion protein is abolished by ectopic expression of prion protein-like protein

This study was designed to examine the function of cellular prion protein and prion protein-like protein/Doppel, in transient ischemia-related neuronal death in the hippocampus. Two different lines of mice devoid of cellular prion protein, Zrch I Prnp(0/0) and Ngsk Prnp(0/0), were used. The former lacks cellular prion protein whereas the latter ectopically expresses prion protein-like protein/Doppel in the brain in the absence of cellular prion protein. Mice were subjected to 10 min-occlusion of the bilateral common carotid arteries with recovery for 14 days. Less than 10% of the pyramidal neurons in the CA1 subfield were degenerated in male and female wild-type mice. In contrast, more than half of the neurons were lost in male Zrch I Prnp(0/0) and Ngsk Prnp(0/0) mice. Such severe neuronal loss was also observed in female Ngsk Prnp(0/0) mice. However, female Zrch I Prnp(0/0) mice showed mild neuronal loss similar to wild-type mice. Flunarizine, a T- and L-type Ca(2+)-channel antagonist, significantly reduced the neuronal loss in female but not in male Ngsk Prnp(0/0) mice. These results indicate that loss of cellular prion protein renders hippocampal neurons susceptible to ischemic insult specifically in male but not female mice and the ectopic expression of prion protein-like protein/Doppel aggravates the ischemic neuronal death in female prion protein-null mice probably via overloading of Ca(2+)-dependent signaling.