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Amyloid fibrils, which are closely associated with various neurodegenerative diseases, are the final products in many protein aggregation pathways. The identification of fibrils at low concentration is, therefore, pivotal in disease diagnosis and development of therapeutic strategies. We report a methodology for the specific identification of amyloid fibrils using chiroptical effects in plasmonic nanoparticles. The formation of amyloid fibrils based on α-synuclein was probed using gold nanorods, which showed no apparent interaction with monomeric proteins but effective adsorption onto fibril structures via noncovalent interactions. The amyloid structure drives a helical nanorod arrangement, resulting in intense optical activity at the surface plasmon resonance wavelengths. This sensing technique was successfully applied to human brain homogenates of patients affected by Parkinson’s disease, wherein protein fibrils related to the disease were identified through chiral signals from Au nanorods in the visible and near IR, whereas healthy brain samples did not exhibit any meaningful optical activity. The technique was additionally extended to the specific detection of infectious amyloids formed by prion proteins, thereby confirming the wide potential of the technique. The intense chiral response driven by strong dipolar coupling in helical Au nanorod arrangements allowed us to detect amyloid fibrils down to nanomolar concentrations.

Prions are thought to be the proteinaceous infectious agents responsible for transmissible spongiform encephalopathies (TSEs). PrPSc, the main component of the infectious agent, is also the only validated surrogate marker for the disease, and its sensitive detection is critical for minimizing the spread of the disease. We detected PrPSc biochemically in the blood of hamsters infected with scrapie during most of the presymptomatic phase of the disease. At early stages of the incubation period, PrPSc detected in blood was likely to be from the peripheral replication of prions, whereas at the symptomatic phase, PrPSc in blood was more likely to have leaked from the brain. The ability to detect prions biochemically in the blood of infected but not clinically sick animals offers a great promise for the noninvasive early diagnosis of TSEs.

Efforts to contain the spread of chronic wasting disease (CWD), a fatal, contagious prion disease of cervids, would be aided by the availability of additional diagnostic tools. RT-QuIC assays allow ultrasensitive detection of prion seeds in a wide variety of cervid tissues, fluids and excreta. The best documented antemortem diagnostic test involving RT-QuIC analysis targets lymphoid tissue in rectal biopsies. Here we have tested a more easily accessed specimen, ear pinna punches, using an improved RT-QuIC assay involving iron oxide magnetic extraction to detect CWD infections in asymptomatic mule and white-tailed deer. Comparison of multiple parts of the ear pinna indicated that a central punch spanning the auricular nerve provided the most consistent detection of CWD infection. When compared to results obtained from gold-standard retropharyngeal lymph node specimens, our RT-QuIC analyses of ear samples provided apparent diagnostic sensitivity (81%) and specificity (91%) that rivaled, or improved upon, those observed in previous analyses of rectal biopsies using RT-QuIC. These results provide evidence that RT-QuIC analysis of ear pinna punches may be a useful approach to detecting CWD infections in cervids.

Prion diseases are a group of rapidly progressing neurodegenerative disorders caused by the misfolding of the endogenous prion protein (PrP C ) into a pathogenic form (PrP Sc ). This process, despite being the central event underlying these disorders, remains largely unknown at a molecular level, precluding the prediction of new potential outbreaks or interspecies transmission incidents. In this work, we present a method to generate bona fide recombinant prions de novo, allowing a comprehensive analysis of protein misfolding across a wide range of prion proteins from mammalian species. We study more than 380 different prion proteins from mammals and classify them according to their spontaneous misfolding propensity and their conformational variability. This study aims to address fundamental questions in the prion research field such as defining infectivity determinants, interspecies transmission barriers or the structural influence of specific amino acids and provide invaluable information for future diagnosis and therapy applications. To study neurodegenerative prion diseases, a method (PMSA) for generating prions spontaneously is presented. Applied to 380+ different prion proteins, their tendency to become pathogenic was ranked, illuminating their formation process.

The role of the glycosylation status of PrP C in the conversion to its pathological counterpart and on cross-species transmission of prion strains has been widely discussed. Here, we assessed the effect on strain characteristics of bovine spongiform encephalopathy (BSE) isolates with different transmission histories upon propagation on a model expressing a non-glycosylated human PrP C . Bovine, ovine and porcine-passaged BSE, and variant Creutzfeldt-Jakob disease (vCJD) isolates were used as seeds/inocula in both in vitro and in vivo propagation assays using the non-glycosylated human PrP C -expressing mouse model (TgNN6h). After protein misfolding cyclic amplification (PMCA), all isolates maintained the biochemical characteristics of BSE. On bioassay, all PMCA-propagated BSE prions were readily transmitted to TgNN6h mice, in agreement with our previous in vitro results. TgNN6h mice reproduced the characteristic neuropathological and biochemical hallmarks of BSE, suggesting that the absence of glycans did not alter the pathobiological features of BSE prions. Moreover, back-passage of TgNN6h-adapted BSE prions to BoTg110 mice recovered the full BSE phenotype, confirming that the glycosylation of human PrP C is not essential for the preservation of the human transmission barrier for BSE prions or for the maintenance of BSE strain properties.

Prions are infectious proteins composed of the abnormal disease-causing isoform PrPSc, which induces conformational conversion of the host-encoded normal cellular prion protein PrPC to additional PrPSc. The mechanism underlying prion strain mutation in the absence of nucleic acids remains unresolved. Additionally, the frequency of strains causing chronic wasting disease (CWD), a burgeoning prion epidemic of cervids, is unknown. Using susceptible transgenic mice, we identified two prevalent CWD strains with divergent biological properties but composed of PrPSc with indistinguishable biochemical characteristics. Although CWD transmissions indicated stable, independent strain propagation by elk PrPC, strain coexistence in the brains of deer and transgenic mice demonstrated unstable strain propagation by deer PrPC. The primary structures of deer and elk prion proteins differ at residue 226, which, in concert with PrPSc conformational compatibility, determines prion strain mutation in these cervids.

Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively by the misfolded prion protein (PrP Sc ). Disease is transmitted by the autocatalytic propagation of PrP Sc misfolding at the expense of the normal prion protein. The biggest challenge of the prion hypothesis has been to explain the molecular mechanism by which prions can exist as different strains, producing diseases with distinguishable characteristics. Here, we show that PrP Sc generated in vitro by protein misfolding cyclic amplification from five different mouse prion strains maintains the strain‐specific properties. Inoculation of wild‐type mice with in vitro ‐generated PrP Sc caused a disease with indistinguishable incubation times as well as neuropathological and biochemical characteristics as the parental strains. Biochemical features were also maintained upon replication of four human prion strains. These results provide additional support for the prion hypothesis and indicate that strain characteristics can be faithfully propagated in the absence of living cells, suggesting that strain variation is dependent on PrP Sc properties.

Prion diseases, particularly sporadic cases, pose a challenge due to their complex nature and heterogeneity. The underlying mechanism of the spontaneous conversion from PrP C to PrP Sc , 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.

Background Inherited prion diseases follow autosomal dominant inheritance with a theoretical 50% transmission risk per pregnancy. Transmission ratio distortion (TRD)—deviation from expected Mendelian ratios—has been documented in other genetic disorders but never systematically studied in prionopathies. We aimed to determine whether TRD occurs in families with inherited prion diseases—specifically familial Creutzfeldt‐Jakob disease due to the p.E200K variant and fatal familial insomnia caused by the p.D178N variant—and to assess whether transmission patterns differ by parent sex. Methods We analyzed 24 pedigrees (12 per variant) comprising 65 nuclear families with 151 offspring. Transmission ratios were calculated and compared to expected 50% Mendelian inheritance using generalized estimating equations. Sex‐specific transmission patterns were evaluated for both maternal and paternal carriers. Results Overall transmission rates were 67.1% for p.D178N (95% CI: 56.3%–77.9%, p < 0.05) and 70.5% for p.E200K (95% CI: 60.4%–80.6%, p < 0.001), representing deviations from expected Mendelian ratios. Sex‐specific analysis revealed maternal transmission of 67.5% for p.D178N (p < 0.05) and 67.3% for p.E200K (p < 0.05). Paternal transmission showed 66.7% for p.D178N (p = 0.056) and 78.3% for p.E200K (p < 0.05), with the latter showing pronounced paternal bias. Conclusions This study provides the first systematic evidence of TRD in inherited prion diseases, with both variants showing clinically meaningful deviations from Mendelian inheritance. These findings challenge current genetic counseling assumptions of 50% inheritance risk and suggest that actual transmission probabilities are 17%–21% higher than traditionally estimated. Risk assessment protocols for prion diseases require updating to incorporate variant‐specific and sex‐specific transmission patterns, particularly the strong paternal bias observed with p.E200K. This study investigated whether transmission ratio distortion (TRD) occurs in inherited prion diseases, specifically familial Creutzfeldt‐Jakob disease (p.E200K variant) and fatal familial insomnia (p.D178N variant), despite the theoretical 50% autosomal dominant inheritance risk. Analysis of 24 pedigrees (65 nuclear families) revealed that overall transmission rates were significantly higher than expected Mendelian ratios. These findings challenge current genetic counseling assumptions of a 50% risk, suggesting that the actual probability of transmission is 17%–21% higher, and emphasize the need to update risk assessment protocols to incorporate variant‐specific and sex‐specific transmission patterns, particularly the pronounced paternal bias observed with the p.E200K variant.

One of the characteristics of prions is their ability to infect some species but not others and prion resistant species have been of special interest because of their potential in deciphering the determinants for susceptibility. Previously, we developed different in vitro and in vivo models to assess the susceptibility of species that were erroneously considered resistant to prion infection, such as members of the Leporidae and Equidae families. Here we undertake in vitro and in vivo approaches to understand the unresolved low prion susceptibility of canids. Studies based on the amino acid sequence of the canine prion protein (PrP), together with a structural analysis in silico, identified unique key amino acids whose characteristics could orchestrate its high resistance to prion disease. Cell- and brain-based PMCA studies were performed highlighting the relevance of the D163 amino acid in proneness to protein misfolding. This was also investigated by the generation of a novel transgenic mouse model carrying this substitution and these mice showed complete resistance to disease despite intracerebral challenge with three different mouse prion strains (RML, 22L and 301C) known to cause disease in wild-type mice. These findings suggest that dog D163 amino acid is primarily, if not totally, responsible for the prion resistance of canids.