Prion diseases, particularly sporadic cases, pose a challenge due to their complex nature and heterogeneity. The underlying mechanism of the spontaneous conversion from PrPC to PrPSc, the hallmark of prion diseases, remains elusive. To shed light on this process and the involvement of cofactors, we have developed an in vitro system that faithfully mimics spontaneous prion misfolding using minimal components. By employing this PMSA methodology and introducing an isoleucine residue at position 108 in mouse PrP, we successfully generated recombinant murine prion strains with distinct biochemical and biological properties. Our study aimed to explore the influence of a polyanionic cofactor in modulating strain selection and infectivity in de novo-generated synthetic prions. These results not only validate PMSA as a robust method for generating diverse bona fide recombinant prions but also emphasize the significance of cofactors in shaping specific prion conformers capable of crossing species barriers. Interestingly, once these conformers are established, our findings suggest that cofactors are not necessary for their infectivity. This research provides valuable insights into the propagation and maintenance of the pathobiological features of cross-species transmissible recombinant murine prion and highlights the intricate interplay between cofactors and prion strain characteristics.The present work was partially funded by three different grants awarded by “Agencia Estatal de Investigación, Ministerio de Ciencia e Innovación” (Spanish Government), grant numbers PID2021-122201OB-C21 granted to J.C., PID2021-1222010B-C22 granted to E.V., and PID2020-117465GB-I00 granted to J.R.R., funded by MCIN/ AEI /10.13039/501100011033 and co-financed by the European Regional Development Fund (ERDF). EFA031/01 NEURO-COOP, which is co-funded at 65% by the European Union through Programa Interreg VI-A España-Francia-Andorra (POCTEFA 2021-2027) granted to J.C. and H.E. Additional funding was provided by the Instituto de Salud Carlos III (ISCIII), grant number AC21_2/00024, granted to J.C. Additionally, CIC bioGUNE currently holds a Severo Ochoa Excellence accreditation, CEX2021-001136-S granted to J.C., also funded by Ministerio de Ciencia e Innovación/AEI /10.13039/501100011033. Transgenic Facility, directed by M.A.S.-M., is supported by Instituto de Salud Carlos III (ISCIII), co-funded by the European Union grant PT23/00123. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio
