D178N prion protein mutation endows RML prions with new strain properties that do not mimic human genetic prion diseases

Genetic prion diseases are caused by mutant prion protein (PrP) misfolding, eventually leading to the formation of PrP^Sc, the infectious prion isoform that propagates by inducing misfolding of native PrP. Different mutations are thought to generate distinct prion strains with unique self-replicating and neurotoxic properties, contributing to the phenotypic diversity of genetic prion diseases. We previously showed that transgenic mice expressing the mouse PrP homologs of the D178N-M129 and D178N-V129 mutations linked to fatal familial insomnia (FFI) and genetic Creutzfeldt-Jakob disease (CJD¹⁷⁸) accumulate misfolded, mildly proteinase-K (PK)-resistant PrP in their brains. These mice develop spontaneous neurological illnesses resembling FFI and CJD¹⁷⁸, but their diseases have not been found to be transmissible to various mouse lines. In this study, we further assessed their prion propagation potential by inoculating bank voles-shown here to be susceptible to human FFI and CJD¹⁷⁸ prions-and by using RT-QuIC. Negative results from both approaches corroborate the idea that these mice do not generate infectious prions. However, when brain homogenates from Tg(FFI) and Tg(CJD) mice were subjected to protein misfolding cyclic amplification with RML PrP^Sc as a seed, they generated highly PK-resistant mutant prions (RML^FFI and RML^CJD) able to propagate in Tga20 mice overexpressing wild-type PrP. To determine whether these in vitro-converted prions modeled the human diseases better, we examined their transmissibility, biochemical traits, and neuropathological features. Despite successful serial propagation in Tga20 mice, RML^FFI and RML^CJD displayed long incubation times, poor transmissibility to C57BL/6 mice, identical PK-resistant PrP fragments, and distinctive neuropathological changes including large submeningeal and perivascular plaques enriched in endogenous proteolytically shed PrP lacking membrane anchorage. These findings indicate that, regardless of the M129V polymorphism, the D178N mutation imparts novel, stable strain properties to RML that do not recapitulate the features of FFI and CJD¹⁷⁸. Our results offer new insights into how genetic PrP mutations influence prion strain characteristics and suggest that spontaneous and templated prionogenesis may follow distinct mechanistic pathways.