Yoshifumi Iwamaru

PrPSc distribution of a natural case of bovine spongiform encephalopathy

Bovine spongiform encephalopathy (BSE) is a disease of cattle that causes progressive neurodegeneration of the central nervous system. Infectivity of BSE agent is accompanied with an abnormal isoform of prion protein (PrPSc).

Proximity of SCG10 and prion protein in membrane rafts.

The conversion of normal cellular prion protein (PrPC) into its pathogenic isoform (PrPSc) is an essential event in prion pathogenesis. In culture models, membrane rafts are suggested to play a critical role in PrPSc formation. To identify the candidate molecules capable of interacting with PrPC and facilitating PrPSc formation in membrane rafts, we applied a novel biochemical labeling method termed enzyme‐mediated activation of radical sources. Enzyme‐mediated activation of radical sources was applied to the Lubrol WX insoluble detergent‐resistant membrane fractions from mouse neuroblastoma (N2a) cells in which the surface PrPC was labeled with HRP‐conjugated anti‐PrP antibody. Two‐dimensional western blots of these preparations revealed biotinylated spots of approximately 20 kDa with an isoelectric point of 8.0–9.0. Liquid chromatography–tandem mass spectrometry analysis resulted in the identification of peptides containing SCG10, the neuron‐specific microtubule regulator. Proximity of SCG10 and PrPC was confirmed using proximity ligation assay and co‐immunoprecipitation assay. Transfection of persistently 22L prion‐infected N2a cells with SCG10 small interfering RNA reduced SCG10 expression, but did not prevent PrPSc accumulation, indicating that SCG10 appears to be unrelated to PrPSc formation of 22L prion. Immunofluorescence and western blot analyses showed reduced levels of SCG10 in the hippocampus of prion‐infected mice, suggesting a possible association between SCG10 levels and the prion neuropathogenesis.

Chronic wasting disease prion infection of differentiated neurospheres

ABSTRACT A possible strategy to develop more diverse cell culture systems permissive to infection with naturally occurring prions is to exploit culture of neurospheres from transgenic mice expressing the normal prion protein (PrP) of the native host species. Accordingly, we developed differentiated neurosphere cultures from the cervid PrP-expressing mice to investigate whether this in vitro system would support replication of non-adapted cervid-origin chronic wasting disease (CWD) prions. Here we report the successful amplification of disease-associated PrP in differentiated neurosphere cultures within 3 weeks after exposure to CWD prions from both white-tailed deer or elk. This neurosphere culture system provides a new in vitro tool that can be used to assess non-adapted CWD prion propagation and transmission.

Role of Autophagy in P2X7 Receptor-Mediated Maturation and Unconventional Secretion of IL-1β in Microglia

Interleukin-1β (IL-1β) is a potent proinflammatory cytokine that is mainly produced by microglia in the central nervous system. It is considered to act as a key mediator of inflammatory processes not only in physiological conditions, but also during various pathological conditions, such as infection, injury, ischemia, and neurodegeneration. The mechanism through which such a leaderless protein is transferred from the cytoplasm to the extracellular space is an important unresolved issue in the study of IL-1β biology. Emerging evidence suggests that autophagy plays an important role in the unconventional secretion of IL-1β. Autophagy might negatively regulate IL-1β expression by lysosomal degradation, while mature IL-1β could be secreted by an autophagy-based Golgi reassembly stacking protein (GRASP)-dependent secretory pathway. We also found that activation of the P2X7 receptor (P2X7R), an ATP-gated cation channel, plays a critical role in the regulation of basal autophagy flux as well as the maturation and unconventional secretion of IL-1β in microglial cells. Taken together, better understanding of the role of the autophagy-lysosomal pathway in the maturation and secretion of IL-1β in microglia might provide a new strategy for targeting neuroinflammation in various pathological conditions.

CHAPTER 15:Role of P2X7 Receptor Signaling in the Treatment of Parkinson’s Disease and Other Neurodegenerative Disorders

Neurodegenerative diseases such as Parkinson’s disease (PD) and prion disease are characterized by protein aggregation and gliosis, including astrocytes and microglia. Currently, there is no treatment for prevention of disease progression. It has been well characterized that neuroinflammation caused by aberrant activation of glial cells may play a critical role in the pathogenesis of neurodegenerative diseases, and thus modulation of neuroinflammatory reactions may be a possible therapeutic strategy. In this context, we focus here on the P2X7 receptor (P2X7R), a cation‐selective ion channel gated by extracellular adenosine triphosphate, as a drug target for the modulation of neuroinflammation in PD. P2X7R is highly expressed in microglia in the central nervous system and has a pivotal role in the maturation and release of the powerful pro‐inflammatory cytokine interleukin‐1β (IL‐1β). Importantly, increased expression of IL‐1β correlates with the progression of PD, and up‐regulation of P2X7R expression is also observed in animal models of PD. Furthermore, evidence is accumulating to indicate that P2X7R may be involved in a variety of cellular events that lead to both neurodegeneration and neuroprotection. Thus, drugs that modulate P2X7R activity may provide a new strategy for treatment of Parkinson’s and other neurodegenerative diseases.

Partial characterization of monoclonal antibodies which bind to disease-associated prion protein in immunoprecipitaion assay

Conformational conversion of cellular prion protein (PrPC) to scrapie-form prion protein (PrPSc) is thought to be the central event in prion pathogenesis. Several studies have shown that their distinct conformation should cause different immunogenic properties, however, almost all mAbs generated against PrP are unable to recognize each ones separately. Such characters of mAbs make it diffcult to study conformational difference between PrPC and PrPSc or mechanism of prion replication. Therefore the purpose of this study is to generate such mAbs that would react with PrPSc but not with PrPC.

Surveillance of chronic wasting disease (CWD) in Japan

Chronic wasting disease (CWD) in cervids including elk, mule deer, and white-tailed deer, is a member of the transmissible spongiform encephalopathies (TSEs). CWD is a serious problem in North America. The detection of abnormal isoforms of prion protein (PrPSc) is a key factor for the diagnosis of CWD, similar to other TSEs. The surveillance program for TSEs in animals is conducted by the Ministry of Agriculture, Forestry, and Fishery (MAFF) and is targeted to sheep, goats, and deer. In Japan, several different anti-prion protein (PrP) monoclonal antibodies (mAbs) are utilized for bovine spongiform encephalopathy (BSE) confirmation. Since CWD does not occur naturally in Japan, the immunoreactivity of the antibodies against PrPSc found in deer was not known. In this study, we examined the immunoreactivities of these antibodies against PrPSc found in CWD. The protocols that are used in Japan for confirmation of BSE cases are Western blot (WB) and immunohistochemistry (IHC). We used these same protocols to examine CWD positive brain samples which were provided by Dr. A. Davis of the National Veterinary Service Laboratory, USA. Mabs. T1, T2, 44B1, and 72-5, were used successfully to detect PrPSc in CWD affected mule deer brains by WB. In IHC, PrPSc was detected with mAbs T2, 44B1, and polyclonal antibody B103. These results determined that the antibodies used for BSE confirmation are also applicable to CWD, as for scrapie. These same antibodies could detect PrPC from Japanese deer by WB without proteinase digestion. The amino acid sequence of PrP of Japanese deer was found to be the same as sequence as the one reported for mule deer. These antibodies were then used for CWD surveillance in Japan. When 127 of hunter-killed deer from Hokkaido were examined, PrPSc was not detected in any of the animals.

Report of the first oral inoculation of BSE prion into cattle in Japan

A Prion Disease Research Center at the National Institute of Animal Health (NIAH) was established to conduct comprehensive research on BSE in response to the discovery of bovine spongiform encephalopathy (BSE) in Japan. A new research facility for the center has been constructed. It was designed as a biosafety level (BSL) 3 facility with the capacity to inoculate and hold experimental animals. Experiments have begun to infect cattle with BSE orally. This route of inoculation simulates the feeding of contaminated meat and bone meal that caused pandemic occurrence of BSE in the UK. An abnormal isoform of the prion protein (PrPSc) accumulates in BSE affected cattle. The purpose of this study is to examine the spread of the abnormal prion from digestive tract to the central nervous system and to describe the pathological changes in cattle during the course of infection. Atypical BSE and young BSE cases have been found in the abattoir surveillance program. As a result the cattle used in this experiment were imported from Australia, a country free of BSE, to exclude the possibility of prior BSE infection before inoculation. Each calf (Holstein heifer, 10-months old) was inoculated orally with 5g of brain stem from BSE infected cattle (courtesy provided by Veterinary Laboratory Agency, Weybridge, UK) into the rumen with a catheter. The cattle will be euthanized at intervals during the clinical stages of disease. Infectivity from different tissues at different stages of clinical disease as well as the deposition of PrPSc will be analyzed. Other factors that may be related to the pathogenesis of the BSE prion will be investigated.