Biology subjects

Amano, G.

Publications and source records attributed to Amano, G..

3 recordsLinked to original sources

bioRxiv2026

PRNP mutations initially generate an alternatively misfolded PrP species that dissuades prion replication

Mutations in the PRNP gene, which encodes the prion protein (PrP), cause genetic prion disease. However, how PRNP mutations lead to spontaneous prion formation remains poorly understood. Building off the observation that expression of mutant bank vole PrP (BVPrP) in mice causes spontaneous prion disease, we sought to identify early events in prion formation by expressing mutant BVPrP in cultured cells lacking endogenous PrP. Expression of D178N- and E200K-mutant, but not wild-type BVPrP, in CAD5-PrP-/- and N2a-PrP-/- cells generates an alternatively misfolded PrP species, which we term PrPAM, that is detergent-insoluble and resistant to digestion with the protease thermolysin. PrPAM is also present in the brains of young, asymptomatic knock-in mice expressing mutant BVPrP. However, PrPAM does not appear to be a direct precursor of prions since 1) the prion disease-protective G127V substitution enhances PrPAM levels when coupled with the D178N or E200K mutations; 2) small molecule anti-prion drugs fail to reduce PrPAM accumulation in cells; 3) unlike cells expressing wild-type BVPrP, cells expressing D178N- or E200K-mutant BVPrP are largely resistant to infection with several prion strains; and 4) PrPAM-containing cell homogenate does not contain seeds that induce misfolding of wild-type BVPrP in either cells or the RT-QuIC assay. These results raise the possibility that prion disease-causing PRNP mutations initially generate a protective misfolded PrP species that is refractory to prion formation, which provides a potential explanation for why genetic prion diseases manifest later in life despite the presence of a mutation from birth.

neuroscience↗

bioRxiv2025

The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrPC Levels and Prion Replication

Prions are misfolded, self-propagating versions of the cellular prion protein (PrPC) that cause invariably fatal transmissible neurodegenerative diseases in humans and animals. Little is known about how prions replicate in the brain, including whether other proteins participate in prion replication in vivo. Several members of the protein disulfide isomerase family have been shown to reside in close spatial proximity to PrPC in cells and mice, implying that they could be involved in prion biogenesis. Here, we show that stable knock-down of the protein disulfide isomerase P4HB (also called PDIA1) in prion-susceptible CAD5 cells reduces PrPC levels and results in lower levels of protease-resistant PrP (PrPres), a marker of infectious prions, following infection with two different prion strains. Partial reduction of P4HB activity using the P4HB-selective inhibitor KSC-34 also decreases PrPC levels in uninfected CAD5 cells whereas treatment of prion-infected CAD5 cells with KSC-34 results in higher levels of PrPres. A proportion of P4HB reaches the cell surface where PrPC is located, and a secreted P4HB variant increases PrPres levels in cells. Collectively, these results suggest that P4HB facilitates prion replication in cells by stabilizing PrPC and potentially acting as a chaperone that directly modulates prion conversion. Thus, targeting P4HB during prion disease may have therapeutic benefit.

neuroscience↗

bioRxiv2024

The Molecular Determinants of a Universal Prion Acceptor

In prion diseases, the species barrier limits the transmission of prions from one species to another. However, cross-species prion transmission is remarkably efficient in bank voles, and this phenomenon can be recapitulated in mice by expression of the bank vole prion protein (BVPrP). The molecular determinants of BVPrPs ability to function as a universal or near-universal acceptor for prions remain incompletely defined. Building on our finding that cultured cells expressing BVPrP can replicate both mouse and hamster prion strains, we conducted a systematic analysis to identify key residues in BVPrP that permit cross-species prion replication. Consistent with previous findings, we demonstrate that residues N155 and N170 of BVPrP, which are absent in mouse PrP but present in hamster PrP, are critical for cross-species prion replication. Additionally, BVPrP residues V112, I139, and M205, which are absent in hamster PrP but present in mouse PrP, are also required to enable replication of both mouse and hamster prions. Unexpectedly, we found that residues E227 and S230 near the C-terminus of BVPrP severely restrict the accumulation of prions following cross-species prion challenge, suggesting that they may have evolved to counteract the inherent propensity of BVPrP to misfold. PrP variants with an enhanced ability to replicate both mouse and hamster prions displayed accelerated spontaneous aggregation kinetics in vitro. These findings suggest that BVPrPs unusual properties are governed by a key set of amino acids and that the enhanced misfolding propensity of BVPrP may enable cross-species prion replication.

neuroscience↗