Daisuke Ishibashi

Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion

The development of technologies for the in vitro amplification of abnormal conformations of prion protein (PrPSc) has generated the potential for sensitive detection of prions. Here we developed a new PrPSc amplification assay, called real-time quaking-induced conversion (RT-QUIC), which allows the detection of ≥1 fg of PrPSc in diluted Creutzfeldt-Jakob disease (CJD) brain homogenate. Moreover, we assessed the technique first in a series of Japanese subjects and then in a blind study of 30 cerebrospinal fluid specimens from Australia, which achieved greater than 80% sensitivity and 100% specificity. These findings indicate the promising enhanced diagnostic capacity of RT-QUIC in the antemortem evaluation of suspected CJD.

FK506 reduces abnormal prion protein through the activation of autolysosomal degradation and prolongs survival in prion-infected mice

Prion diseases are fatal neurodegenerative disorders and no effective treatment has been established to date. In this study, we evaluated the effect of FK506 (tacrolimus), a macrolide that is known to be a mild immunosuppressant, on prion infection, using cell culture and animal models. We found that FK506 markedly reduced the abnormal form of prion protein (PRNPSc) in the cell cultures (N2a58 and MG20) infected with Fukuoka-1 prion. The levels of autophagy-related molecules such as LC3-II, ATG12–ATG5 and ATG7 were significantly increased in the FK506-treated cells, and resulted in the increased formation of autolysosomes. Upregulation of the autophagy-related molecules was also seen in the brains of FK506-treated mice and the accumulation of PRNPSc was delayed. The survival periods in mice inoculated with Fukuoka-1 were significantly increased when FK506 was administered from day 20 post-inoculation. These findings provide evidence that FK506 could constitute a novel antiprion drug, capable of enhancing the degradation of PRNPSc in addition to attenuation of microgliosis and neuroprotection.

Increased expression of p62/SQSTM1 in prion diseases and its association with pathogenic prion protein

Prion diseases are neurodegenerative disorders characterized by the aggregation of abnormally folded prion protein (PrPSc). In this study, we focused on the mechanism of clearance of PrPSc, which remains unclear. p62 is a cytosolic protein known to mediate both the formation and degradation of aggregates of abnormal proteins. The levels of p62 protein increased in prion-infected brains and persistently infected cell cultures. Upon proteasome inhibition, p62 co-localized with PrPSc, forming a large aggregate in the perinuclear region, hereafter referred to as PrPSc-aggresome. These aggregates were surrounded with autophagosome marker LC3 and lysosomes in prion-infected cells. Moreover, transient expression of the phosphomimic form of p62, which has enhanced ubiquitin-binding activity, reduced the amount of PrPSc in prion-infected cells, indicating that the activation of p62 could accelerate the clearance of PrPSc. Our findings would thus suggest that p62 could be a target for the therapeutic control of prion diseases.

Hyperefficient PrP Sc amplification of mouse-adapted BSE and scrapie strain by protein misfolding cyclic amplification technique.

Abnormal forms of prion protein (PrPSc) accumulate via structural conversion of normal PrP (PrPC) in the progression of transmissible spongiform encephalopathy. Under cell‐free conditions, the process can be efficiently replicated using in vitro PrPSc amplification methods, including protein misfolding cyclic amplification. These methods enable ultrasensitive detection of PrPSc; however, there remain difficulties in utilizing them in practice. For example, to date, several rounds of protein misfolding cyclic amplification have been necessary to reach maximal sensitivity, which not only take several weeks, but also result in an increased risk of contamination. In this study, we sought to further promote the rate of PrPSc amplification in the protein misfolding cyclic amplification technique using mouse transmissible spongiform encephalopathy models infected with either mouse‐adapted bovine spongiform encephalopathy or mouse‐adapted scrapie, Chandler strain. Here, we demonstrate that appropriate regulation of sonication dramatically accelerates PrPSc amplification in both strains. In fact, we reached maximum sensitivity, allowing the ultrasensitive detection of < 1 LD50 of PrPSc in the diluted brain homogenates, after only one or two reaction rounds, and in addition, we detected PrPSc in the plasma of mouse‐adapted bovine spongiform encephalopathy‐infected mice. We believe that these results will advance the establishment of a fast, ultrasensitive diagnostic test for transmissible spongiform encephalopathies.

Post-ischemic Treatment with Toki-Shakuyaku-San (Tang-Gui-Shao-Yao-San) Prevents the Impairment of Spatial Memory Induced by Repeated Cerebral Ischemia in Rats

Previously we have reported that Toki-shakuyaku-san (TSS) ameliorated the impairment of spatial memory induced by single cerebral ischemia (1 x 10 minutes) and scopolamine, a muscarinic receptor antagonist. In this experiment, we studied the effect of TSS on repeated cerebral ischemia (2 x 10 minutes, 1-hour interval) induced impairment of spatial memory and neuronal injury in rats. The 8-day post-ischemic treatment with TSS (30-300 mg/kg) was administered p.o. once per day. TSS dose-dependently prevented the impairment of spatial memory, neuronal death and TUNEL positive cells induced by repeated cerebral ischemia. In order to determine the mechanism of TSS, we also studied the effect of TSS on GluR2 mRNA, one of the glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) receptor subunits. Repeated cerebral ischemia significantly decreased GluR2 flop mRNA at 1 and 3 days after the occlusion. TSS (300 mg/kg) significantly suppressed the decrease in GluR2 flop at 3 days after repeated cerebral ischemia. These results suggested that the TSS has neuroprotective action which may be indirectly mediated by the AMPA receptor, and TSS may be beneficial for the treatment of cerebrovascular dementia.

Protective Role of Interferon Regulatory Factor 3-Mediated Signaling against Prion Infection

ABSTRACT Abnormal prion protein (PrPSc) generated from the cellular isoform of PrP (PrPC) is assumed to be the main or sole component of the pathogen, called prion, of transmissible spongiform encephalopathies (TSE). Because PrP is a host-encoded protein, acquired immune responses are not induced in TSE. Meanwhile, activation of the innate immune system has been suggested to partially block the progression of TSE; however, the mechanism is not well understood. To further elucidate the role of the innate immune system in prion infection, we investigated the function of interferon regulatory factor 3 (IRF3), a key transcription factor of the MyD88-independent type I interferon (IFN) production pathway. We found that IRF3-deficient mice exhibited significantly earlier onset with three murine TSE strains, namely, 22L, FK-1, and murine bovine spongiform encephalopathy (mBSE), following intraperitoneal transmission, than with wild-type controls. Moreover, overexpression of IRF3 attenuated prion infection in the cell culture system, while PrPSc was increased in prion-infected cells treated with small interfering RNAs (siRNAs) against IRF3, suggesting that IRF3 negatively regulates PrPSc formation. Our findings provide new insight into the role of the host innate immune system in the pathogenesis of prion diseases.

Ubiquitin-specific protease 14 modulates degradation of cellular prion protein.

Prion diseases are fatal neurodegenerative disorders characterized by the accumulation of prion protein (PrPC). To date, there is no effective treatment for the disease. The accumulated PrP, termed PrPSc, forms amyloid fibrils and could be infectious. It has been suggested that PrPSc is abnormally folded and resistant to proteolytic degradation, and also inhibits proteasomal functions in infected cells, thereby inducing neuronal death. Recent work indicates that the ubiquitin-proteasome system is involved in quality control of PrPC. To reveal the significance of prion protein ubiqitination, we focused on ubiquitin-specific protease 14 (USP14), a deubiqutinating enzyme that catalyzes trimming of polyubiquitin chains and plays a role in regulation of proteasomal processes. Results from the present study showed that treatment with a selective inhibitor of USP14 reduced PrPC, as well as PrPSc, levels in prion-infected neuronal cells. Overexpression of the dominant negative mutant form of USP14 reduced PrPSc, whereas wildtype USP14 increased PrPSc in prion-infected cells. These results suggest that USP14 prevents degradation of both normal and abnormal PrP. Collectively, a better understanding about the regulation of PrPSc clearance caused by USP14 might contribute greatly to the development of therapeutic strategies for prion diseases.

Conformational Properties of Prion Strains Can Be Transmitted to Recombinant Prion Protein Fibrils in Real-Time Quaking-Induced Conversion

ABSTRACT The phenomenon of prion strains with distinct biological characteristics has been hypothesized to be involved in the structural diversity of abnormal prion protein (PrPSc). However, the molecular basis of the transmission of strain properties remains poorly understood. Real-time quaking-induced conversion (RT-QUIC) is a cell-free system that uses Escherichia coli-derived recombinant PrP (rPrP) for the sensitive detection of PrPSc. To investigate whether the properties of various prion strains can be transmitted to amyloid fibrils consisting of rPrP (rPrP fibrils) using RT-QUIC, we examined the secondary structure, conformational stability, and infectivity of rPrP fibrils seeded with PrPSc derived from either the Chandler or the 22L strain. In the first round of the reaction, there were differences in the secondary structures, especially in bands attributed to β-sheets, as determined by infrared spectroscopy, and conformational stability between Chandler-seeded (1st-rPrP-fibCh) and 22L-seeded (1st-rPrP-fib22L) rPrP fibrils. Of note, specific identifying characteristics of the two rPrP fibril types seen in the β-sheets resembled those of the original PrPSc. Furthermore, the conformational stability of 1st-rPrP-fibCh was significantly higher than that of 1st-rPrP-fib22L, as with Chandler and 22L PrPSc. The survival periods of mice inoculated with 1st-rPrP-fibCh or 1st-rPrP-fib22L were significantly shorter than those of mice inoculated with mixtures from the mock 1st-round RT-QUIC procedure. In contrast, these biochemical characteristics were no longer evident in subsequent rounds, suggesting that nonspecific uninfected rPrP fibrils became predominant probably because of their high growth rate. Together, these findings show that at least some strain-specific conformational properties can be transmitted to rPrP fibrils and unknown cofactors or environmental conditions may be required for further conservation. IMPORTANCE The phenomenon of prion strains with distinct biological characteristics is assumed to result from the conformational variations in the abnormal prion protein (PrPSc). However, important questions remain about the mechanistic relationship between the conformational differences and the strain diversity, including how strain-specific conformations are transmitted. In this study, we investigated whether the properties of diverse prion strains can be transmitted to amyloid fibrils consisting of E. coli-derived recombinant PrP (rPrP) generated by real-time quaking-induced conversion (RT-QUIC), a recently developed in vitro PrPSc formation method. We demonstrate that at least some of the strain-specific conformational properties can be transmitted to rPrP fibrils in the first round of RT-QUIC by examining the secondary structure, conformational stability, and infectivity of rPrP fibrils seeded with PrPSc derived from either the Chandler or the 22L prion strain. We believe that these findings will advance our understanding of the conformational basis underlying prion strain diversity.

Dominant-negative Effects of the N-terminal Half of Prion Protein on Neurotoxicity of Prion Protein-like Protein/Doppel in Mice

Prion protein-like protein/doppel is neurotoxic, causing ataxia and Purkinje cell degeneration in mice, whereas prion protein antagonizes doppel-induced neurodegeneration. Doppel is homologous to the C-terminal half of prion protein but lacks the amino acid sequences corresponding to the N-terminal half of prion protein. We show here that transgenic mice expressing a fusion protein consisting of the N-terminal half, corresponding to residues 1-124, of prion protein and doppel in neurons failed to develop any neurological signs for up to 730 days in a background devoid of prion protein. In addition, the fusion protein prolonged the onset of ataxia in mice expressing exogenous doppel. These results suggested that the N-terminal part of prion protein has a neuroprotective potential acting both cis and trans on doppel. We also show that prion protein lacking the pre-octapeptide repeat (Δ25-50) or octapeptide repeat (Δ51-90) region alone could not impair the antagonistic function against doppel.

Structure-Based Drug Discovery for Prion Disease Using a Novel Binding Simulation

The accumulation of abnormal prion protein (PrPSc) converted from the normal cellular isoform of PrP (PrPC) is assumed to induce pathogenesis in prion diseases. Therefore, drug discovery studies for these diseases have focused on the protein conversion process. We used a structure-based drug discovery algorithm (termed Nagasaki University Docking Engine: NUDE) that ran on an intensive supercomputer with a graphic-processing unit to identify several compounds with anti-prion effects. Among the candidates showing a high-binding score, the compounds exhibited direct interaction with recombinant PrP in vitro, and drastically reduced PrPSc and protein-aggresomes in the prion-infected cells. The fragment molecular orbital calculation showed that the van der Waals interaction played a key role in PrPC binding as the intermolecular interaction mode. Furthermore, PrPSc accumulation and microgliosis were significantly reduced in the brains of treated mice, suggesting that the drug candidates provided protection from prion disease, although further in vivo tests are needed to confirm these findings. This NUDE-based structure-based drug discovery for normal protein structures is likely useful for the development of drugs to treat other conformational disorders, such as Alzheimers disease.