Liquid-liquid phase separation of full-length prion protein initiates conformational conversion in vitro.

Prion diseases are characterized by the accumulation of amyloid fibrils. The causative agent is an infectious amyloid that comprises solely misfolded prion protein (PrPSc). Prions can convert normal cellular prion protein (PrPC) to protease K-resistance prion protein fragment (PrP-res) in vitro; however, the intermediate steps involved in this spontaneous conversion still remain unknown. We investigated whether recombinant prion protein (rPrP) can directly convert into PrP-res via liquid-liquid phase separation (LLPS) in the absence of PrPSc. We found that rPrP underwent LLPS at the interface of the aqueous two-phase system of polyethylene glycol and dextran, whereas single-phase conditions were not inducible. Fluorescence recovery assay after photobleaching revealed that the liquid-solid phase transition occurred within a short time. The aged rPrP-gel acquired a proteinase-resistant amyloid accompanied by ß-sheet conversion, as confirmed by Western blotting, Fourier transform infrared spectroscopy, and Congo red staining. The reactions required both the N-terminal region of rPrP (amino acids 23-89) and kosmotropic salts, suggesting that the kosmotropic anions may interact with the N-terminal region of rPrP to promote LLPS. Thus, structural conversion via LLPS and liquid-solid phase transition could be the intermediate steps in the conversion of prions.

Dextran sulphate inhibits an association of prions with plasma membrane at the early phase of infection.

The defining characteristic of prion diseases is conversion of a cellular prion protein (PrPC) to an abnormal prion protein (PrPSc). The exogenous attachment of PrPSc to the surface of a target cell is critical for infection. However, the initial interaction of PrPSc with the cell surface is poorly characterized. In the current study, we specifically focused on the association of PrPSc with cells during the early phase of infection, using an acute infection model. First, we treated mouse neuroblastoma N2a-58 cells with prion strain 22 L-infected brain homogenates and revealed that PrPSc was associated with membrane fractions within three hours, a short exposure time. These results were also observed in PrPC-deficient hippocampus cell lines. We also demonstrate here that PrPSc from 22 L-infected brain homogenates was associated with lipid rafts during the early phase of infection. Furthermore, we revealed that DS500, a glycosaminoglycan mimetic, inhibited both the attachment of PrPSc to membrane fractions and subsequent prion transmission, suggesting that the early association of prions with cell surface is important for prion infection.

Novel Compounds Identified by Structure-Based Prion Disease Drug Discovery Using In Silico Screening Delay the Progression of an Illness in Prion-Infected Mice.

The accumulation of abnormal prion protein (PrPSc) produced by the structure conversion of PrP (PrPC) in the brain induces prion disease. Although the conversion process of the protein is still not fully elucidated, it has been known that the intramolecular chemical bridging in the most fragile pocket of PrP, known as the "hot spot," stabilizes the structure of PrPC and inhibits the conversion process. Using our original structure-based drug discovery algorithm, we identified the low molecular weight compounds that predicted binding to the hot spot. NPR-130 and NPR-162 strongly bound to recombinant PrP in vitro, and fragment molecular orbital (FMO) analysis indicated that the high affinity of those candidates to the PrP is largely dependent on nonpolar interactions, such as van der Waals interactions. Those NPRs showed not only significant reduction of the PrPSc levels but also remarkable decrease of the number of aggresomes in persistently prion-infected cells. Intriguingly, treatment with those candidate compounds significantly prolonged the survival period of prion-infected mice and suppressed prion disease-specific pathological damage, such as vacuole degeneration, PrPSc accumulation, microgliosis, and astrogliosis in the brain, suggesting their possible clinical use. Our results indicate that in silico drug discovery using NUDE/DEGIMA may be widely useful to identify candidate compounds that effectively stabilize the protein.

Administration of FK506 from Late Stage of Disease Prolongs Survival of Human Prion-Inoculated Mice.

Human prion diseases are etiologically categorized into three forms: sporadic, genetic, and infectious. Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common type of human prion disease that manifests as subacute progressive dementia. No effective therapy for sCJD is currently available. Potential therapeutic compounds are frequently tested in rodents infected with mouse-adapted prions that differ from human prions. However, therapeutic effect varies depending on the prion strain, which is one of the reasons why candidate compounds have shown little effect in sCJD patients. We previously reported that intraperitoneal administration of FK506 was able to prolong the survival of mice infected with a mouse-adapted prion by suppressing the accumulation of abnormal prion protein (PrP) and inhibiting the activation of microglia. In this study, we tested oral administration of FK506 in knock-in mice expressing chimeric human prion protein (KiChM) that were infected with sCJD to determine if this compound is also effective against a clinically relevant human prion, i.e., one that has not been adapted to mice. Treatment with FK506, started either just before or just after disease onset, suppressed typical sCJD pathology (gliosis) and slightly but significantly prolonged the survival of sCJD-inoculated mice. It would be worthwhile to conduct a clinical trial using FK506, which has been safety-approved and is widely used as a mild immunosuppressant.