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.

The prion-like seeding of misfolded α-synuclein (αSyn) involved in the pathogenesis of Lewy body diseases (LBD) remains poorly understood at the molecular level. Using the real-time quaking-induced conversion (RT-QUIC) seeding assay, we investigated whether brain tissues from cases of dementia with Lewy bodies (DLB), which contain serine 129 (Ser129)-phosphorylated insoluble aggregates of αSyn, can convert Escherichia coli-derived recombinant αSyn (r-αSyn) to fibrils. Diffuse neocortical DLB yielded 50% seeding dose (SD₅₀) values of 10⁷~10¹⁰/g brain. Limbic DLB was estimated to have an SD₅₀ value of ~10⁵/g brain. Furthermore, RT-QUIC assay discriminated DLB from other neurological and neurodegenerative disorders. Unexpectedly, the prion-like seeding was reconstructed in reactions seeded with oligomer-like species, but not with insoluble aggregates of r-αSyn, regardless of Ser129 phosphorylation status. Our findings suggest that RT-QUIC using r-αSyn can be applied to detect seeding activity in LBD, and the culprit that causes prion-like seeding may be oligomeric forms of αSyn.

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.

Prion diseases are fatal neurodegenerative disorders characterized by the accumulation of prion protein (PrP^C). To date, there is no effective treatment for the disease. The accumulated PrP, termed PrP^Sc, forms amyloid fibrils and could be infectious. It has been suggested that PrP^Sc 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 PrP^C. 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 PrP^C, as well as PrP^Sc, levels in prion-infected neuronal cells. Overexpression of the dominant negative mutant form of USP14 reduced PrP^Sc, whereas wildtype USP14 increased PrP^Sc 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 PrP^Sc clearance caused by USP14 might contribute greatly to the development of therapeutic strategies for prion diseases.

The accumulation of abnormal prion protein (PrP(Sc)) converted from the normal cellular isoform of PrP (PrP(C)) 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 PrP(Sc) 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 PrP(C) binding as the intermolecular interaction mode. Furthermore, PrP(Sc) 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 Alzheimer's disease.