Prion Protein Research Articles

PrPC Glycoprotein Is Indispensable for Maintenance of Skeletal Muscle Homeostasis During Aging.

The cellular prion protein (PrPC), a glycoprotein encoded by the PRNP gene, is known to modulate muscle mass and exercise capacity. However, the role of PrPC in the maintenance and regeneration of skeletal muscle during ageing remains unclear. This study investigated the change in PrPC expression during muscle formation using C2C12 cells and evaluated muscle function in Prnp wild-type (WT) and knock-out (KO) mice at different ages (1, 9 and 15 months). To determine the role of PrPC in skeletal muscle homeostasis during ageing, we conducted regeneration experiments via cardiotoxin injection in Prnp mice to assess the effects of PrPC deficiency on the senescence of satellite stem cells (SCs) and regenerative capacity in skeletal muscle. Our data demonstrate that PrPC expression increased significantly during muscle differentiation (p < 0.01), correlating with myogenin (immunofluorescence at the differentiation stage). PrPC deficiency disrupted muscle homeostasis, leading to age-associated mitochondrial autophagy (Pink-1, +180%, p < 0.001; Parkin, +161%, p < 0.01) and endoplasmic reticulum stress (SERCA, -26%, p < 0.05; IRE1α, +195%, p < 0.001) while decreasing the level of mitochondrial biogenesis (SIRT-1, -50%, p < 0.01; PGC-1α, -36%, p < 0.05; VDAC, -27%, p < 0.001), and activated oxidative stress (serum myoglobin, +23%, p < 0.001; MDA, +23%, p < 0.05; NFκB, +117%, p < 0.05) during ageing, which accelerated reduced muscle growth or mass accumulation (tibialis anterior muscle mass, -23%, p < 0.001; gastrocnemius muscle mass, -30%, p < 0.001; muscle fibre size, -48%, p < 0.05; MSTN, +160%, p < 0.01; MAFbx, +83%, p < 0.05). Furthermore, PrPC deficiency induced the senescence (β-galactosidase, +60%, p < 0.05; p16, +103%, p < 0.001) of SCs, which was directly related to the defect in muscle recovery, with the senescence-mediated enhancement of adipogenesis (PPARγ, +74%, p < 0.05) during the regeneration process after cardiotoxin-induced muscle injury. Our findings demonstrate that PrPC is indispensable for maintaining skeletal muscle homeostasis during ageing by modulating the functional integrity of mitochondria, ER and SCs.

Exploring immunotherapeutic strategies for neurodegenerative diseases: a focus on Huntington's disease and Prion diseases.

Immunotherapy has emerged as a promising therapeutic approach for the treatment of neurodegenerative disorders, which are characterized by the progressive loss of neurons and impaired cognitive functions. In this review, active and passive immunotherapeutic strategies that help address the underlying pathophysiology of Huntington's disease (HD) and prion diseases by modulating the immune system are discussed. The current landscape of immunotherapeutic strategies, including monoclonal antibodies and vaccine-based approaches, to treat these diseases is highlighted, along with their potential benefits and mechanisms of action. Immunotherapy generally works by targeting disease-specific proteins, which serve as the pathological hallmarks of these diseases. Additionally, the review addresses the challenges and limitations associated with immunotherapy. For HD, immunotherapeutic approaches focus on neutralizing the toxic effects of mutant huntingtin and tau proteins, thereby reducing neurotoxicity. Immunotherapeutic approaches targeting flanking sequences, rather than the polyglutamine tract in the mutant huntingtin protein, have yielded promising outcomes for patients with HD. In prion diseases, therapies attempt to prevent or eliminate misfolded proteins that cause neurodegeneration. The major challenge in prion diseases is immune tolerance. Approaches to overcome the highly tolerogenic nature of the prion protein have been discussed. A common hurdle in delivering antibodies is the blood‒brain barrier, and strategies that can breach this barrier are being investigated. As protein aggregation and neurotoxicity are related, immunotherapeutic strategies being developed for other neurodegenerative diseases could be repurposed to target protein aggregation in HD and prion diseases. While significant advances in this field have been achieved, continued research and development are necessary to overcome the existing limitations, which will help in shaping the future of immunotherapy as a strategy for managing neurological disorders.

Multiomic analyses direct hypotheses for Creutzfeldt-Jakob disease risk genes.

Prions are assemblies of misfolded prion protein that cause several fatal and transmissible neurodegenerative diseases, with the most common phenotype in humans being sporadic Creutzfeldt-Jakob disease (sCJD). Aside from variation of the prion protein itself, molecular risk factors are not well understood. Prion and prion-like mechanisms are thought to underpin common neurodegenerative disorders meaning that the elucidation of mechanisms could have broad relevance. Herein we sought to further develop our understanding of the factors that confer risk of sCJD using a systematic gene prioritization and functional interpretation pipeline based on multiomic integrative analyses. We integrated the published sCJD genome-wide association study (GWAS) summary statistics with publicly available bulk brain and brain cell type gene and protein expression datasets. We performed multiple transcriptome and proteome-wide association studies (TWAS & PWAS) and Bayesian genetic colocalization analyses between sCJD risk association signals and multiple brain molecular quantitative trait loci signals. We then applied our systematic gene prioritization pipeline on the obtained results and nominated prioritized sCJD risk genes with risk-associated molecular mechanisms in a transcriptome and proteome-wide manner. Genetic upregulation of both gene and protein expression of syntaxin-6 (STX6) in the brain was associated with sCJD risk in multiple datasets, with a risk-associated gene expression regulation specific to oligodendrocytes. Similarly, increased gene and protein expression of protein disulfide isomerase family A member 4 (PDIA4), involved in the unfolded protein response, was linked to increased disease risk, particularly in excitatory neurons. Protein expression of mesencephalic astrocyte derived neurotrophic factor (MANF), involved in protection against endoplasmic reticulum stress and sulfatide binding (linking to the enzyme in the final step of sulfatide synthesis, encoded by sCJD risk gene GAL3ST1), was identified as protective against sCJD. In total 32 genes were prioritized into two tiers based on the level of evidence and confidence for further studies. This study provides insights into the genetically-associated molecular mechanisms underlying sCJD susceptibility and prioritizes several specific hypotheses for exploration beyond the prion protein itself and beyond the previously highlighted sCJD risk loci through the newly prioritized sCJD risk genes and mechanisms. These findings highlight the importance of glial cells, sulfatides and the excitatory neuron unfolded protein response in sCJD pathogenesis.

Tau oligomers impair memory and synaptic plasticity through the cellular prion protein

Deposition of abnormally phosphorylated tau aggregates is a central event leading to neuronal dysfunction and death in Alzheimer’s disease (AD) and other tauopathies. Among tau aggregates, oligomers (TauOs) are considered the most toxic. AD brains show significant increase in TauOs compared to healthy controls, their concentration correlating with the severity of cognitive deficits and disease progression. In vitro and in vivo neuronal TauO exposure leads to synaptic and cognitive dysfunction, but their mechanisms of action are unclear. Evidence suggests that the cellular prion protein (PrPC) may act as a mediator of TauO neurotoxicity, as previously proposed for β-amyloid and α-synuclein oligomers. To investigate whether PrPC mediates TauO detrimental activities, we compared their effects on memory and synaptic plasticity in wild type (WT) and PrPC knockout (Prnp0/0) mice. Intracerebroventricular injection of TauOs significantly impaired recognition memory in WT but not in Prnp0/0 mice. Similarly, TauOs inhibited long-term potentiation in acute hippocampal slices from WT but not Prnp0/0 mice. Surface plasmon resonance indicated a high-affinity binding between TauOs and PrPC with a KD of 20–50 nM. Immunofluorescence analysis of naïve and PrPC-overexpressing HEK293 cells exposed to TauOs showed a PrPC dose-dependent association of TauOs with cells over time, and their co-localization with PrPC on the plasma membrane and in intracellular compartments, suggesting PrPC-may play a role in TauO internalization. These findings support the concept that PrPC mediates the detrimental activities of TauOs through a direct interaction, suggesting that targeting this interaction might be a promising therapeutic strategy for AD and other tauopathies.

Prion protein modulation of virus-specific T cell differentiation and function during acute viral infection.

The differentiation and functionality of virus-specific T cells during acute viral infections are crucial for establishing long-term protective immunity. While numerous molecular regulators impacting T cell responses have been uncovered, the role of cellular prion proteins (PrPc) remains underexplored. Here, we investigated the impact of PrPc deficiency on the differentiation and function of virus-specific T cells using the lymphocytic choriomeningitis virus (LCMV) Armstrong acute infection model. Our findings reveal that Prnp-/- mice exhibit a robust expansion of virus-specific CD8+ T cells, with similar activation profiles as wild-type mice during the early stages of infection. However, Prnp-/- mice had higher frequencies and numbers of virus-specific memory CD8+ T cells, along with altered differentiation profiles characterized by increased central and effector memory subsets. Despite similar proliferation rates early during infection, Prnp-/- memory CD8+ T cells had decreased proliferation compared with their wild-type counterparts. Additionally, Prnp-/- mice had higher numbers of cytokine-producing memory CD8+ T cells, indicating a more robust functional response. Furthermore, Prnp-/- mice had increased virus-specific CD4+ T cell responses, suggesting a broader impact of PrPc deficiency on T cell immunity. These results unveil a previously unrecognized role for PrPc in regulating the differentiation, proliferation, and functionality of virus-specific T cells, providing valuable insights into immune system regulation by prion proteins during viral infections.

Unlocking Neuroinflammation: A Balanced Art for Therapeutics of Prion Disease.

Neuroinflammation plays a dual role in prion diseases, contributing both to the clearance of misfolded scrapie-like prion protein and to neuropathology through chronic activation of inflammatory pathways. Key mechanisms, including M-CSF/CSF1R signaling, NLRP3 inflammasome activation, and the Galectin-3/TREM2 axis, etc., highlight the complexity of targeting neuroinflammation for therapeutic intervention. Balancing the modulation of these pathways while preserving beneficial immune responses and maintaining blood-brain barrier integrity is critical for developing effective treatments.

Chaperone-Mediated Heterotypic Phase Separation Prevents the Amyloid Formation of the Pathological Y145Stop Prion Protein Variant.

Biomolecular condensates formed via phase separation of proteins and nucleic acids are crucial for the spatiotemporal regulation of a diverse array of essential cellular functions and the maintenance of cellular homeostasis. However, aberrant liquid-to-solid phase transitions of such condensates are associated with several fatal human diseases. Such dynamic membraneless compartments can contain a range of molecular chaperones that can regulate the phase behavior of proteins involved in the formation of these biological condensates. Here, we show that a heat shock protein 40 (Hsp40), Ydj1, exhibits a holdase activity by potentiating the phase separation of a disease-associated stop codon mutant of the prion protein (Y145Stop) either by recruitment into Y145Stop condensates or via Y145Stop-Ydj1 two-component heterotypic phase separation that arrests the conformational conversion of Y145Stop into amyloid fibrils. Utilizing site-directed mutagenesis, multicolor fluorescence imaging, single-droplet steady-state and picosecond time-resolved fluorescence anisotropy, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy, we delineate the complex network of interactions that govern the heterotypic phase separation of Y145Stop and Ydj1. We also show that the properties of such heterotypic condensates can further be tuned by RNA that promotes the formation of multicomponent multiphasic protein-RNA condensates. Our vibrational Raman spectroscopy results in conjunction with atomic force microscopy imaging reveal that Ydj1 effectively redirects the self-assembly of Y145Stop towards a dynamically-arrested non-amyloidogenic pathway, preventing the formation of typical amyloid fibrils. Our findings underscore the importance of chaperone-mediated heterotypic phase separation in regulating aberrant phase transitions and amyloid formation associated with a wide range of deadly neurodegenerative diseases.

Identification of the Highly Polymorphic Prion Protein Gene (PRNP) in Frogs (Rana dybowskii).

Prion diseases are fatal neurodegenerative diseases that can be transmitted by infectious protein particles, PrPScs, encoded by the endogenous prion protein gene (PRNP). The origin of prion seeds is unclear, especially in non-human hosts, and this identification is pivotal to preventing the spread of prion diseases from host animals. Recently, an abnormally high amyloid propensity in prion proteins (PrPs) was found in a frog, of which the genetic variations in the PRNP gene have not been investigated. In this study, genetic polymorphisms in the PRNP gene were investigated in 194 Dybowski's frogs using polymerase chain reaction (PCR) and amplicon sequencing. We carried out in silico analyses to predict functional alterations according to non-synonymous single nucleotide polymorphisms (SNPs) using PolyPhen-2, PANTHER, SIFT, and MutPred2. We used ClustalW2 and MEGA X to compare frog PRNP and PrP sequences with those of prion-related animals. To evaluate the impact of the SNPs on protein aggregation propensity and 3D structure, we utilized AMYCO and ColabFold. We identified 34 novel genetic polymorphisms including 6 non-synonymous SNPs in the frog PRNP gene. The hydrogen bond length varied at codons 143 and 207 according to non-synonymous SNPs, even if the electrostatic potential was not changed. In silico analysis predicted S143N to increase the aggregation propensity, and W6L, C8Y, R211W, and L241F had damaging effects on frog PrPs. The PRNP and PrP sequences of frogs showed low homology with those of prion-related mammals. To the best of our knowledge, this study was the first to discover genetic polymorphisms in the PRNP gene in amphibians.

In vivo base editing extends lifespan of a humanized mouse model of prion disease.

Prion disease is a fatal neurodegenerative disease caused by the misfolding of prion protein (PrP) encoded by the PRNP gene. While there is currently no cure for the disease, depleting PrP in the brain is an established strategy to prevent or stall templated misfolding of PrP. Here we developed in vivo cytosine and adenine base strategies delivered by adeno-associated viruses to permanently modify the PRNP locus to achieve PrP knockdown in the mouse brain. Systemic injection of dual-adeno-associated virus PHP.eB encoding BE3.9max and single guide RNA installing PRNP R37X resulted in 37% average installation of the desired edit, 50% reduction of PrP in the mouse brain and 52% extension of lifespan in transgenic human PRNP mice inoculated with pathogenic human prion isolates representing the most common sporadic and genetic subtypes of prion disease. We further engineered base editing systems to achieve improved in vivo potency and reduced base editor expression in nontargeting tissues, resulting in 63% average PrP reduction in the mouse brain from a 6.7-fold lower viral dose, with no detected off-target editing of anticipated clinical significance observed in either human cells or mouse tissues. These findings support the potential of in vivo base editing as one-time treatment for prion disease.

The Knowns and Unknowns of Prion Protein in Immune Modulation and the Pathogenesis of Neuroautoimmune Diseases

The Knowns and Unknowns of Prion Protein in Immune Modulation and the Pathogenesis of Neuroautoimmune Diseases

Demyelinating neuropathy as the initial presentation of familial E200K Creutzfeldt-Jakob disease in two patients.

To describe peripheral neuropathy associated with familial Creutzfeldt-Jakob disease. We report two unrelated patients with genetic Creutzfeldt-Jakob disease with demyelinating peripheral neuropathy as initial presentation, with a comprehensive clinical, electrophysiological and neuropathological description. Both patients exhibited gait disturbance and paresthesia. Electrodiagnostic studies revealed demyelinating abnormalities with motor conduction blocks suggestive of chronic inflammatory demyelinating polyradiculoneuropathy, with abnormal plexus MRI and elevated CSF protein levels. One of them had pes cavus and a late-onset Charcot-Marie-Tooth (CMT) disease was also initially hypothesized. Central nervous system involvement manifested 1-2 years after the onset of peripheral symptoms. Both patients had a heterozygous E200K mutation in the PRNP gene. Postmortem neuropathological examinations showed PrPSc deposits in the peripheral nervous system, particularly in Schwann cells. Peripheral neuropathy in E200K genetic forms of Creutzfeldt-Jakob disease can be inaugural and mimic chronic inflammatory demyelinating polyradiculoneuropathy.

The brain interactome of a permissive prion replication substrate.

Bank voles are susceptible to prion strains from many different species, yet the molecular mechanisms underlying the ability of bank vole prion protein (BVPrP) to function as a universal prion acceptor remain unclear. Potential differences in molecular environments and protein interaction networks on the cell surface of brain cells may contribute to BVPrP's unusual behavior. To test this hypothesis, we generated knock-in mice that express physiological levels of BVPrP (M109 isoform) and employed mass spectrometry to compare the interactomes of mouse (Mo) PrP and BVPrP following mild in vivo crosslinking of brain tissue. Substantial overlap was observed between the top interactors for BVPrP and MoPrP, with established PrP-interactors such as neural cell adhesion molecules, subunits of Na+/K+-ATPases, and contactin-1 being equally present in the two interactomes. We conclude that the molecular environments of BVPrP and MoPrP in the brains of mice are very similar. This suggests that the unorthodox properties of BVPrP are unlikely to be mediated by differential interactions with other proteins.

The novel T107I Inherited prion disease can present as a clinical and biomarker mimic of familial Alzheimer’s disease

Inherited prion diseases (IPD) secondary to mutations of the prion protein gene, PRNP, exhibit diverse clinical phenotypes, capable of mimicking numerous primary neurodegenerative conditions. We describe the clinical phenotype and neuropathological findings in a family from County Limerick in Ireland presenting with Alzheimer’s disease-like cognitive decline and motor symptoms caused by a novel missense mutation of PRNP. This mutation occurs in the PRNP central lysine cluster (CLC; codon 101-110), resulting in substitution of threonine with isoleucine at codon 107 (T107I). This case series highlights that IPD can be hard to distinguish from overlapping clinical syndromes seen in other neurodegenerative diseases. We also discuss similarities and differences of the novel mutation T107I to other pathogenic mutations of the CLC of PRNP.

Comparing the Extent of Methionine Oxidation in the Prion and Native Conformations of PrP.

Scrapie is a prion disease of sheep and goats. Prions (PrPSc) replicate by inducing a natively expressed protein (PrPC) to refold into the prion conformation. PrPC and PrPSc contain a disproportionately large number of methionines. Surface exposed methionines are more prone to chemical oxidation. Chemical oxidation is a means of measuring the surface exposure of the methionines in a prion, as these covalent changes are retained after an oxidized prion is denatured prior to analysis. Scrapie prions and recombinant sheep prion protein were oxidized in 0, 10, 20, or 50 mM solutions of hydrogen peroxide. The samples were digested with trypsin or trypsin followed by chymotrypsin to yield a set of peptides (TNMK, MLGSAMSR, ENMYR, IMER, VVEQMCITQYQR) containing the methionines present in sheep PrP. The mass spectrometry based multiple reaction monitoring (MRM) method was used to analyze these peptides. Analysis of the rPrP samples showed that surface exposed methionines (132, 137, and 157) were more oxidized than those less surface exposed (209 and 216). The extent of methionine oxidation in sheep scrapie PrPSc is 216 > 137 > 132 > 157 > 209 > 112. These results demonstrate that this approach can be used to map the surface exposure of the methionines in order to distinguish among PrP conformations and effect a kind of conformational sequence.

Preanalytical Considerations of Handling Suspected Creutzfeldt-Jakob Disease Specimens Within the Clinical Pathology Laboratories: A Survey-Based Approach.

Background: Creutzfeldt-Jakob disease (CJD) is a rare, fatal, and transmissible neurodegenerative disorder caused by prion proteins. Handling specimens from individuals with suspected or confirmed cases presents a safety challenge to hospital workers including clinical laboratory staff. As no national guidelines exist, the clinical pathology laboratory must establish protocols for handling these specimens to ensure sufficient protective measures. This study aims to explore how various medical institutions manage CJD specimens, as a first step toward developing standardized preanalytical protocols for safe specimen handling by health care professionals. Methods: An electronic survey was generated and disseminated to diplomats of the American Board of Clinical Chemistry and was posted on the Listserv platform of the American Society for Microbiology and the Artery forum of the Association for Diagnostics and Laboratory Medicine. The survey evaluated various procedures and precautions implemented, the nature of the specimens processed, and whether they are processed in-house or sent to reference laboratories. Results: A total of 49 responses were collected. Most respondents (64%) noted their laboratories process specimens with a clinical suspicion of CJD regardless of the level of suspicion, 13% handled specimens only if the degree of suspicion was low, and 16% did not process specimens in-house at all. Among those who process CJD specimens, practices varied greatly, including different levels of precautions, use of biological safety cabinets, aliquoting, disposal, and disinfection procedures. Conclusions: A lack of standardization across laboratories exists for the handling of specimens of patients with suspected CJD. This study summarizes the approaches reported by survey respondents, providing a rationale for developing protocols for the safe handling of these specimens and highlighting the need to develop uniform universal standardized processing procedures.

Prion Protein Endoproteolysis: Cleavage Sites, Mechanisms and Connections to Prion Disease.

Highly abundant in neurons, the cellular prion protein (PrPC) is an obligatory precursor to the disease-associated misfolded isoform denoted PrPSc that accumulates in the rare neurodegenerative disorders referred to either as transmissible spongiform encephalopathies (TSEs) or as prion diseases. The ability of PrPC to serve as a substrate for this template-mediated conversion process depends on several criteria but importantly includes the presence or absence of certain endoproteolytic events performed at the cell surface or in acidic endolysosomal compartments. The major endoproteolytic events affecting PrPC are referred to as α- and β-cleavages, and in this review we outline the sites within PrPC at which the cleavages occur, the mechanisms potentially responsible and their relevance to pathology. Although the association of α-cleavage with neuroprotection is well-supported, we identify open questions regarding the importance of β-cleavage in TSEs and suggest experimental approaches that could provide clarification. We also combine findings from invitro cleavage assays and mass spectrometry-based studies of prion protein fragments in the brain to present an updated view in which α- and β-cleavages may represent two distinct clusters of proteolytic events that occur at multiple neighbouring sites rather than at single positions. Furthermore, we highlight the candidate proteolytic mechanisms best supported by the literature; currently, despite several proteases identified as capable of processing PrPC invitro, in cell-based models and in some cases, invivo, none have been shown conclusively to cleave PrPC in the brain. Addressing this knowledge gap will be crucial for developing therapeutic interventions to drive PrPC endoproteolysis in a neuroprotective direction. Finally, we end this review by briefly addressing other cleavage events, specifically ectodomain shedding, γ-cleavage, the generation of atypical pathological fragments in the familial prion disorder Gerstmann-Sträussler-Scheinker syndrome and the possibility of an additional form of endoproteolysis close to the PrPC N-terminus.

Chaperone-mediated disaggregation of infectious prions releases particles that seed new prion formation in a strain-specific manner.

The mammalian prion protein can form infectious, nonnative, and protease resistant aggregates (PrPD), which cause lethal prion diseases like human Creutzfeldt-Jakob disease. PrPD seeds the formation of new infectious prions by interacting with and triggering the refolding of the normally soluble mammalian prion protein, PrPC, into more PrPD. Refolding of misfolded proteins in the cell is carried out by molecular chaperones such as Grp78. We have recently shown that Grp78 sensitizes PrPD to proteases, indicating structural alterations and leading to its degradation. However, the process of chaperone-mediated PrPD disaggregation, the chaperones involved, and the effect of disaggregation on PrPD seeding activity are unclear. We have now monitored the structural modification, disaggregation, and seeding activity of PrPD from twomouse adapted prion strains, 22L and 87V, in the presence of Grp78 and two forms of the Hsp110 disaggregase chaperone family, Hsp105 and Apg-2. We found that both forms of Hsp110 induced similar amounts of disaggregation and structural change in the protease resistant cores of PrPD from both strains. However, 22L PrPD was more susceptible to destabilization and disaggregation by the chaperones than 87V. Surprisingly, despite disaggregation of both strains, only the 22L PrPD aggregates released by the chaperones had seeding activity, with both forms of Hsp110 enhancing the Grp78 mediated release of these aggregates. Our data show that disassembly of PrPD by Grp78 and Hsp110 chaperones can release seeding particles of PrPD in a strain-specific manner, potentially facilitating prion replication and spread.

Novel method for classification of prion diseases by detecting PrPres signal patterns from formalin-fixed paraffin-embedded samples

ABSTRACT Prion disease is an infectious and fatal neurodegenerative disease. Western blotting (WB)-based identification of proteinase K (PK)-resistant prion protein (PrPres) is considered a definitive diagnosis of prion diseases. In this study, we aimed to detect PrPres using formalin-fixed paraffin-embedded (FFPE) specimens from cases of sporadic Creutzfeldt–Jakob disease (sCJD), Gerstmann–Sträussler–Scheinker disease (GSS), glycosylphosphatidylinositol-anchorless prion disease (GPIALP), and V180I CJD. FFPE samples were prepared after formic acid treatment to inactivate infectivity. After deparaffinization, PK digestion was performed, and the protein was extracted. In sCJD, a pronounced PrPres signal was observed, with antibodies specific for type 1 and type 2 PrPres exhibited a strong or weak signals depending on the case. Histological examination of serial sections revealed that the histological changes were compatible with the biochemical characteristics. In GSS and GPIALP, prion protein core-specific antibodies presented as PrPres bands at 8–9 kDa and smear bands, respectively. However, an antibody specific for the C-terminus presented as smears in GSS, with no PrPres detected in GPIALP. It was difficult to detect PrPres in V180I CJD. Collectively, our findings demonstrate the possibility of detecting PrPres in FFPE and classifying the prion disease types. This approach facilitates histopathological and biochemical evaluation in the same sample and is safe owing to the inactivation of infectivity. Therefore, it may be valuable for the diagnosis and research of prion diseases.

A Copper-Binding Peptide with Therapeutic Potential against Alzheimer's Disease: From the Blood-Brain Barrier to Metal Competition.

Alzheimer's disease (AD) is the most common form of dementia worldwide. AD brains are characterized by the accumulation of amyloid-β peptides (Aβ) that bind Cu2+ and have been associated with several neurotoxic mechanisms. Although the use of copper chelators to prevent the formation of Cu2+-Aβ complexes has been proposed as a therapeutic strategy, recent studies show that copper is an important neuromodulator that is essential for a neuroprotective mechanism mediated by Cu2+ binding to the cellular prion protein (PrPC). Therefore, in addition to metal selectivity and blood-brain barrier (BBB) permeability, an emerging challenge for copper chelators is to prevent the formation of neurotoxic Cu2+-Aβ species without perturbing the neuroprotective Cu2+-PrPC interaction. Previously, we reported the design of a tetrapeptide (TP) that withdraws Cu2+ from Aβ(1-16) and impacts the Cu2+-induced aggregation of Aβ(1-40). In this study, we improved the drug-like properties of TP in a BBB model, evaluated the metal selectivity of the optimized peptide (TP*), and tested its effect on Cu2+ coordination to PrPC and proteins involved in copper trafficking, such as copper transporter 1 and albumin. Our results show that changing the stereochemistry of the first residue prevents TP degradation in the BBB model and coadministration of TP with a peptide that increases BBB permeability allows its passage through the BBB model. TP* is highly selective toward Cu2+ in the presence of Zn2+ ions, transfers Cu2+ to copper-trafficking proteins, and forms a ternary TP*-Cu2+-PrP species that does not perturb the physiological conformation of PrP and displays only a minor impact in the neuroprotective Cu2+-dependent interaction of PrPC with the N-methyl-d-aspartate receptor. Overall, these results show that TP* displays desirable features for a copper chelator with therapeutic potential against AD. Moreover, this is the first study that explores the effect of a Cu2+ chelator with therapeutic potential for AD on Cu2+ coordination to PrPC (an emerging key player in AD pathology), integrating recent knowledge about metalloproteins involved in AD with the design of copper chelators against AD.

RAPID GENOTYPING OF PRNP GENE POLYMORPHISMS TO ASSESS SCRAPIE SUSCEPTIBILITY IN BRAZILIAN SHEEP

Objective: Our study aimed to develop and validate a low-density SNP panel for 15 PRNP gene polymorphisms for genotyping sheep from 15 breeds in Brazil, incorporating samples from the Brazilian Animal Germplasm Biobank (BBGA). It was estimated both allelic, genotypic and haplotypic frequencies for the selected markers. Theoretical Framework: The study hypothesis centers on the need to validate a cost-effective reduced SNP panel, enhance genotyping accuracy, and support selective breeding against scrapie in Brazilian sheep. Method: The methodology adopted comprises a reduced SNP panel, with markers associated with scrapie, designed to genotype DNA samples of Brazilian sheep breeds. Results and Discussion: The panel achieved a success rate of 73%, as out of the 15 genotyped markers, only four were excluded by the call rate. Moreover, 73% (845) of the initial 1,152 samples were successfully genotyped. Analyzing the three most important PRNP polymorphisms, we found that genotype ARQ/ARQ, associated with susceptibility to scrapie, was the most frequent in all breeds. In contrast, genotype ARR/ARR (resistance) was found only in the BBGA (4%). Research Implications: Overall, the low-density SNP panel can be a rapid, dependable, and cost-effective tool for enhancing genetic selection in Brazilian sheep. However, further technology optimization is necessary, which can be achieved by excluding and replacing non-functional markers. Originality/Value: This study presents valuable insights by providing a cost-effective, validated genotyping tool tailored to SNP markers associated with scrapie in Brazilian sheep, which can streamline genetic selection in breeding programs.