Rie Hasebe

Effect of intraventricular infusion of anti-prion protein monoclonal antibodies on disease progression in prion-infected mice

It is well known that anti-prion protein (PrP) monoclonal antibodies (mAbs) inhibit abnormal isoform PrP (PrPSc) formation in cell culture. Additionally, passive immunization of anti-PrP mAbs protects the animals from prion infection via peripheral challenge when mAbs are administered simultaneously or soon after prion inoculation. Thus, anti-PrP mAbs are candidates for the treatment of prion diseases. However, the effects of mAbs on disease progression in the middle and late stages of the disease remain unclear. This study carried out intraventricular infusion of mAbs into prion-infected mice before and after clinical onset to assess their ability to delay disease progression. A 4-week infusion of anti-PrP mAbs initiated at 120 days post-inoculation (p.i.), which is just after clinical onset, reduced PrPSc levels to 70-80 % of those found in mice treated with a negative-control mAb. Spongiform changes, microglial activation and astrogliosis in the hippocampus and thalamus appeared milder in mice treated with anti-PrP mAbs than in those treated with a negative-control mAb. Treatment with anti-PrP mAb prolonged the survival of mice infected with Chandler or Obihiro strain when infusion was initiated at 60 days p.i., at which point PrPSc is detectable in the brain. In contrast, infusion initiated after clinical onset prolonged the survival time by about 8 % only in mice infected with the Chandler strain. Although the effects on survival varied for different prion strains, the anti-PrP mAb could partly prevent disease progression, even after clinical onset, suggesting immunotherapy as a candidate for treatment of prion diseases.

Characterization of intracellular localization of PrP Sc in prion-infected cells using a mAb that recognizes the region consisting of aa 119-127 of mouse PrP

Generation of an abnormal isoform of the prion protein (PrP(Sc)) is a key aspect of the propagation of prions. Elucidation of the intracellular localization of PrP(Sc) in prion-infected cells facilitates the understanding of the cellular mechanism of prion propagation. However, technical improvement in PrP(Sc)-specific detection is required for precise analysis. Here, we show that the mAb 132, which recognizes the region adjacent to the most amyloidogenic region of PrP, is useful for PrP(Sc)-specific detection by immunofluorescence assay in cells pre-treated with guanidine thiocyanate. Extensive analysis of the intracellular localization of PrP(Sc) in prion-infected cells using mAb 132 revealed the presence of PrP(Sc) throughout endocytic compartments. In particular, some of the granular PrP(Sc) signals that were clustered at peri-nuclear regions appeared to be localized in an endocytic recycling compartment through which exogenously loaded transferrin, shiga and cholera toxin B subunits were transported. The granular PrP(Sc) signals at peri-nuclear regions were dispersed to the peripheral regions including the plasma membrane during incubation at 20 °C, at which temperature transport from the plasma membrane to peri-nuclear regions was impaired. Conversely, dispersed PrP(Sc) signals appeared to return to peri-nuclear regions within 30 min during subsequent incubation at 37 °C, following which PrP(Sc) at peri-nuclear regions appeared to redisperse again to peripheral regions over the next 30 min incubation. These results suggest that PrP(Sc) is dynamically transported along with the membrane trafficking machinery of cells and that at least some PrP(Sc) circulates between peri-nuclear and peripheral regions including the plasma membrane via an endocytic recycling pathway.

Equine major histocompatibility complex class I molecules act as entry receptors that bind to equine herpesvirus-1 glycoprotein D

The endotheliotropism of equine herpesvirus‐1 (EHV‐1) leads to encephalomyelitis secondary to vasculitis and thrombosis in the infected horse central nervous system (CNS). To identify the host factors involved in EHV‐1 infection of CNS endothelial cells, we performed functional cloning using an equine brain microvascular endothelial cell cDNA library. Exogenous expression of equine major histocompatibility complex (MHC) class I heavy chain genes conferred susceptibility to EHV‐1 infection in mouse NIH3T3 cells, which are not naturally susceptible to EHV‐1 infection. Equine MHC class I molecules bound to EHV‐1 glycoprotein D (gD), and both anti‐gD antibodies and a soluble form of gD blocked viral entry into NIH3T3 cells stably expressing the equine MHC class I heavy chain gene (3T3‐A68 cells). Treatment with an anti‐equine MHC class I monoclonal antibody blocked EHV‐1 entry into 3T3‐A68 cells, equine dermis (E. Derm) cells and equine brain microvascular endothelial cells. In addition, inhibition of cell surface expression of MHC class I molecules in E. Derm cells drastically reduced their susceptibility to EHV‐1 infection. These results suggest that equine MHC class I is a functional gD receptor that plays a pivotal role in EHV‐1 entry into equine cells.

Infectious entry of equine herpesvirus-1 into host cells through different endocytic pathways.

Abstract We investigated the mechanism by which equine herpesvirus-1 (EHV-1) enters primary cultured equine brain microvascular endothelial cells (EBMECs) and equine dermis (E. Derm) cells. EHV-1 colocalized with caveolin in EBMECs and the infection was greatly reduced by the expression of a dominant negative form of equine caveolin-1 (ecavY14F), suggesting that EHV-1 enters EBMECs via caveolar endocytosis. EHV-1 entry into E. Derm cells was significantly reduced by ATP depletion and treatments with lysosomotropic agents. Enveloped virions were detected from E. Derm cells by infectious virus recovery assay after viral internalization, suggesting that EHV-1 enters E. Derm cells via energy- and pH-dependent endocytosis. These results suggest that EHV-1 utilizes multiple endocytic pathways in different cell types to establish productive infection.

Effect of Transplantation of Bone Marrow-Derived Mesenchymal Stem Cells on Mice Infected with Prions

ABSTRACT Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to migrate to brain lesions in experimental models of ischemia, tumors, and neurodegenerative diseases and to ameliorate functional deficits. In this study, we attempted to evaluate the therapeutic potential of MSCs for treating prion diseases. Immortalized human MSCs (hMSCs) that express the LacZ gene were transplanted into the unilateral hippocampi or thalami of mice, and their distributions were monitored by the expression of β-galactosidase. In mice infected with prions, hMSCs transplanted at 120 days postinoculation (dpi) were detected on the contralateral side at 2 days after transplantation and existed there even at 3 weeks after transplantation. In contrast, few hMSCs were detected on the contralateral side for mock-infected mice. Interestingly, the migration of hMSCs appeared to correlate with the severity of neuropathological lesions, including disease-specific prion protein deposition. The hMSCs also migrated to a prion-specific lesion in the brain, even when intravenously injected. Although the effects were modest, intrahippocampal and intravenous transplantation of hMSCs prolonged the survival of mice infected with prions. A subpopulation of hMSCs in the brains of prion-infected mice produced various trophic factors and differentiated into cells of neuronal and glial lineages. These results suggest that MSCs have promise as a cellular vehicle for the delivery of therapeutic genes to brain lesions associated with prion diseases and, furthermore, that they may help to regenerate neuronal tissues damaged by prion propagation.

Transcellular transport of West Nile virus-like particles across human endothelial cells depends on residues 156 and 159 of envelope protein

BackgroundWest Nile virus (WNV) causes viremia after invasion to the hosts by mosquito bite. Endothelial cells could play an important role in WNV spread from the blood stream into the central nervous system and peripheral tissues. Here, we analyzed the capacity of virus-like particles (VLPs) of the highly virulent NY99 6-LP strain (6-LP VLPs) and the low virulence Eg101 strain (Eg VLPs) to cross cultured human endothelial cells.Results6-LP VLPs were transported from the apical to basolateral side of endothelial cells, whereas Eg VLPs were hardly transported. The localization of tight junction marker ZO-1 and the integrity of tight junctions were not impaired during the transport of 6-LP VLPs. The transport of 6-LP VLPs was inhibited by treatment with filipin, which prevents the formation of cholesterol-dependent membrane rafts, suggesting the involvement of raft-associated membrane transport. To determine the amino acid residues responsible for the transport of VLPs, we produced mutant VLPs, in which residues of E protein were exchanged between the 6-LP and Eg strains. Double amino acid substitution of the residues 156 and 159 greatly impaired the transport of VLPs.ConclusionOur results suggest that a transcellular pathway is associated with 6-LP VLPs transport. We also showed that the combination of the residues 156 and 159 plays an important role in the transport of VLPs across endothelial cells.

Absence of CD14 Delays Progression of Prion Diseases Accompanied by Increased Microglial Activation

ABSTRACT Prion diseases are fatal neurodegenerative disorders characterized by accumulation of PrPSc, vacuolation of neurons and neuropil, astrocytosis, and microglial activation. Upregulation of gene expressions of innate immunity-related factors, including complement factors and CD14, is observed in the brains of mice infected with prions even in the early stage of infections. When CD14 knockout (CD14−/−) mice were infected intracerebrally with the Chandler and Obihiro prion strains, the mice survived longer than wild-type (WT) mice, suggesting that CD14 influences the progression of the prion disease. Immunofluorescence staining that can distinguish normal prion protein from the disease-specific form of prion protein (PrPSc) revealed that deposition of PrPSc was delayed in CD14−/− mice compared with WT mice by the middle stage of the infection. Immunohistochemical staining with Iba1, a marker for activated microglia, showed an increased microglial activation in prion-infected CD14−/− mice compared to WT mice. Interestingly, accompanied by the increased microglial activation, anti-inflammatory cytokines interleukin-10 (IL-10) and transforming growth factor β (TGF-β) appeared to be expressed earlier in prion-infected CD14−/− mice. In contrast, IL-1β expression appeared to be reduced in the CD14−/− mice in the early stage of infection. Double immunofluorescence staining demonstrated that CD11b- and Iba1-positive microglia mainly produced the anti-inflammatory cytokines, suggesting anti-inflammatory status of microglia in the CD14−/− mice in the early stage of infection. These results imply that CD14 plays a role in the disease progression by suppressing anti-inflammatory responses in the brain in the early stage of infection.

The Region Approximately between Amino Acids 81 and 137 of Proteinase K-Resistant PrPSc Is Critical for the Infectivity of the Chandler Prion Strain

ABSTRACT Although the major component of the prion is believed to be the oligomer of PrPSc, little information is available concerning regions on the PrPSc molecule that affect prion infectivity. During the analysis of PrPSc molecules from various prion strains, we found that PrPSc of the Chandler strain showed a unique property in the conformational-stability assay, and this property appeared to be useful for studying the relationship between regions of the PrPSc molecule and prion infectivity. Thus, we analyzed PrPSc of the Chandler strain in detail and analyzed the infectivities of the N-terminally denatured and truncated forms of proteinase K-resistant PrP. The N-terminal region of PrPSc of the Chandler strain showed region-dependent resistance to guanidine hydrochloride (GdnHCl) treatment. The region approximately between amino acids (aa) 81 and 137 began to be denatured by treatment with 1.5 M GdnHCl. Within this stretch, the region comprising approximately aa 81 to 90 was denatured almost completely by 2 M GdnHCl. Furthermore, the region approximately between aa 90 and 137 was denatured completely by 3 M GdnHCl. However, the C-terminal region thereafter was extremely resistant to the GdnHCl treatment. This property was not observed in PrPSc molecules of other prion strains. Denaturation of the region between aa 81 and 137 by 3 M GdnHCl significantly prolonged the incubation periods in mice compared to that for the untreated control. More strikingly, the denaturation and removal of this region nearly abolished the infectivity. This finding suggests that the conformation of the region between aa 81 and 137 of the Chandler strain PrPSc molecule is directly associated with prion infectivity.

Characterization of intracellular dynamics of inoculated PrP-res and newly generated PrPSc during early stage prion infection in Neuro2a cells

To clarify the cellular mechanisms for the establishment of prion infection, we analyzed the intracellular dynamics of inoculated and newly generated abnormal isoform of prion protein (PrP(Sc)) in Neuro2a cells. Within 24h after inoculation, the newly generated PrP(Sc) was evident at the plasma membrane, in early endosomes, and in late endosomes, but this PrP(Sc) was barely evident in lysosomes; in contrast, the majority of the inoculated PrP(Sc) was evident in late endosomes and lysosomes. However, during the subsequent 48 h, the newly generated PrP(Sc) increased remarkably in early endosomes and recycling endosomes. Overexpression of wild-type and mutant Rab proteins showed that membrane trafficking along not only the endocytic-recycling pathway but also the endo-lysosomal pathway is involved in de novo PrP(Sc) generation. These results suggest that the trafficking of exogenously introduced PrP(Sc) from the endo-lysosomal pathway to the endocytic-recycling pathway is important for the establishment of prion infection.

Autophagy inhibits viral genome replication and gene expression stages in West Nile virus infection

Autophagy is a lysosomal degradation pathway that is implicated in many viral infections. However, its role in West Nile virus (WNV) infection remains controversial. In the present study, we examined the relationship between WNV infection and autophagy in infected cells. We demonstrated that LC3-II expression, a molecular marker for autophagosomal membranes, was enhanced in WNV-infected cells 6h post-infection. LC3-II expression was further enhanced in WNV-inoculated cells when treated with a lysosomal protease inhibitor. Meanwhile, WNV replication in cells lacking Atg5, an essential factor for autophagy, was increased compared with replication in wild-type cells. In addition, WNV replication was inhibited in cells lacking Atg5 when they were transfected with an ATG5 expression plasmid. These results suggest an antiviral role for autophagy in WNV-infected cells. We also examined which viral replication stages were affected by autophagy by using a Tat-beclin 1 peptide to induce autophagy and pseudo-infectious WNV reporter virus particles (WNV-RVPs) that monitor viral genome replication and gene expression stages via GFP expression. We found that autophagy induction in HeLa cells by Tat-beclin 1 peptide 3h after WNV inoculation inhibited viral replication, and GFP expression was significantly inhibited in wild-type cells when compared with cells lacking Atg5. Taken together, these results suggest that autophagy is induced by WNV infection, and that this induction inhibits WNV replication at the viral genome replication and gene expression stages.