Creatine Promotes Endometriosis by Inducing Ferroptosis Resistance via Suppression of PrP.

Endometriosis, a chronic inflammatory disease, significantly impairs the quality of life of women in their reproductive years; however, its pathogenesis remains poorly understood. The accumulation of retrograde menstruation and recurrent bleeding fosters a high-iron environment in ectopic lesions, triggering ferroptosis in ectopic endometrial stromal cells (EESCs), thereby hindering the establishment of endometriosis. However, abnormal EESCs demonstrate resistance to ferroptosis in high-iron environments, promoting the progression of this disease. Here, novel findings on the accumulation of creatine, derived from endogenous synthesis, in both peritoneal fluid and EESCs of patients with endometriosis are presented. Creatine supplementation reduces cellular iron concentrations, mitigating oxidative stress and lipid peroxidation, thereby enhancing cell viability and preventing ferroptosis under high-iron conditions. Utilizing the drug affinity-responsive target stabilization (DARTS) assay, prion protein (PrP) as a potential creatine-sensing protein is identified. Mechanistically, creatine binds to the active site of PrP, inhibits the conversion of trivalent iron to divalent iron, and decreases iron uptake, promoting the tolerance of EESCs to ferroptosis. This interaction contributes to the development of endometriosis. The novel association between creatine and ferroptosis provides valuable insights into the role of creatine in endometriosis progression and highlights its potential as a therapeutic target for endometriosis.

Fast-track neuropathological screening for neurodegenerative diseases.

Background: The postmortem diagnostic of individuals having suffered presumptive neurodegenerative disease comprises exclusion of a prion disease, extensive brain sampling and histopathological evaluation, which are resource-intensive and time consuming. To exclude prion disease and to achieve prompt accurate preliminary diagnosis, we developed a fast-track procedure for the histopathological assessment of brains from patients with suspected neurodegenerative disease. Methods: Based on the screening of two brain regions (frontal cortex and cerebellum) with H&E and six immunohistochemical stainings in 133 brain donors, a main histopathological diagnosis was established and compared to the final diagnosis made after a full histopathological work-up according to our brain bank standard procedure. Results: In over 96 % of cases there was a concordance between the fast-track and the final main neuropathological diagnosis. A prion disease was identified in four cases without prior clinical suspicion of a prion infection. Conclusion: The fast-track screening approach relying on two defined, easily accessible brain regions is sufficient to obtain a reliable tentative main diagnosis in individuals with neurodegenerative disease and thus allows for a prompt feedback to the physicians. However, a more thorough histological work-up taking into account the clinical history and the working diagnosis from fast-track screening is necessary for accurate staging and for assessment of co-pathologies.

Lack of prion transmission barrier in human PrP transgenic Drosophila.

While animal prion diseases are a threat to human health, their zoonotic potential is generally inefficient because of interspecies prion transmission barriers. New animal models are required to provide an understanding of these prion transmission barriers and to assess the zoonotic potential of animal prion diseases. To address this goal, we generated Drosophila transgenic for human or non-human primate PrP and determined their susceptibility to known pathogenic prion diseases, namely vCJD and classical BSE, and that with unknown pathogenic potential, namely CWD. Adult Drosophila transgenic for M129 or V129 human PrP, or non-human primate PrP developed a neurotoxic phenotype and showed an accelerated loss of survival after exposure to vCJD, classical BSE, or CWD prions at the larval stage. vCJD prion strain identity was retained after passage in both M129 and V129 human PrP Drosophila. All of the primate PrP fly lines accumulated prion seeding activity and concomitantly developed a neurotoxic phenotype, generally including accelerated loss of survival, after exposure to CWD prions derived from different cervid species, including North American white-tailed deer and muntjac, and European reindeer and moose. These novel studies show that primate PrP transgenic Drosophila lack known prion transmission barriers since, in mammalian hosts, V129 human PrP is associated with severe resistance to classical BSE prions, while both human and cynomolgus macaque PrP are associated with resistance to CWD prions. Significantly, our data suggest that interspecies differences in the amino acid sequence of PrP may not be a principal determinant of the prion transmission barrier.

Current insights and assumptions on α-synuclein in Lewy body disease.

Lewy body disorders are heterogeneous neurological conditions defined by intracellular inclusions composed of misshapen α-synuclein protein aggregates. Although α-synuclein aggregates are only one component of inclusions and not strictly coupled to neurodegeneration, evidence suggests they seed the propagation of Lewy pathology within and across cells. Genetic mutations, genomic multiplications, and sequence polymorphisms of the gene encoding α-synuclein are also causally linked to Lewy body disease. In nonfamilial cases of Lewy body disease, the disease trigger remains unidentified but may range from industrial/agricultural toxicants and natural sources of poisons to microbial pathogens. Perhaps due to these peripheral exposures, Lewy inclusions appear at early disease stages in brain regions connected with cranial nerves I and X, which interface with inhaled and ingested environmental elements in the nasal or gastrointestinal cavities. Irrespective of its identity, a stealthy disease trigger most likely shifts soluble α-synuclein (directly or indirectly) into insoluble, cross-ß-sheet aggregates. Indeed, ß-sheet-rich self-replicating α-synuclein multimers reside in patient plasma, cerebrospinal fluid, and other tissues, and can be subjected to α-synuclein seed amplification assays. Thus, clinicians should be able to capitalize on α-synuclein seed amplification assays to stratify patients into potential responders versus non-responders in future clinical trials of α-synuclein targeted therapies. Here, we briefly review the current understanding of α-synuclein in Lewy body disease and speculate on pathophysiological processes underlying the potential transmission of α-synucleinopathy across the neuraxis.

Multiple aspects of amyloid dynamics in vivo integrate to establish prion variant dominance in yeast.

Prion variants are self-perpetuating conformers of a single protein that assemble into amyloid fibers and confer unique phenotypic states. Multiple prion variants can arise, particularly in response to changing environments, and interact within an organism. These interactions are often competitive, with one variant establishing phenotypic dominance over the others. This dominance has been linked to the competition for non-prion state protein, which must be converted to the prion state via a nucleated polymerization mechanism. However, the intrinsic rates of conversion, determined by the conformation of the variant, cannot explain prion variant dominance, suggesting a more complex interaction. Using the yeast prion system [PSI+ ], we have determined the mechanism of dominance of the [PSI+ ]Strong variant over the [PSI+ ]Weak variant in vivo. When mixed by mating, phenotypic dominance is established in zygotes, but the two variants persist and co-exist in the lineage descended from this cell. [PSI+ ]Strong propagons, the heritable unit, are amplified at the expense of [PSI+ ]Weak propagons, through the efficient conversion of soluble Sup35 protein, as revealed by fluorescence photobleaching experiments employing variant-specific mutants of Sup35. This competition, however, is highly sensitive to the fragmentation of [PSI+ ]Strong amyloid fibers, with even transient inhibition of the fragmentation catalyst Hsp104 promoting amplification of [PSI+ ]Weak propagons. Reducing the number of [PSI+ ]Strong propagons prior to mating, similarly promotes [PSI+ ]Weak amplification and conversion of soluble Sup35, indicating that template number and conversion efficiency combine to determine dominance. Thus, prion variant dominance is not an absolute hierarchy but rather an outcome arising from the dynamic interplay between unique protein conformations and their interactions with distinct cellular proteostatic niches.

Slow Misfolding of a Molten Globule form of a Mutant Prion Protein Variant into a ß-rich Dimer.

Misfolding of the prion protein is linked to multiple neurodegenerative diseases. A better understanding of the process requires the identification and structural characterization of intermediate conformations via which misfolding proceeds. In this study, three conserved aromatic residues (Tyr168, Phe174, and Tyr217) located in the C-terminal domain of mouse PrP (wt moPrP) were mutated to Ala. The resultant mutant protein, 3A moPrP, is shown to adopt a molten globule (MG)-like native conformation. Hydrogen-deuterium exchange studies coupled with mass spectrometry revealed that for 3A moPrP, the free energy gap between the MG-like native conformation and misfolding-prone partially unfolded forms is reduced. Consequently, 3A moPrP misfolds in native conditions even in the absence of salt, unlike wt moPrP, which requires the addition of salt to misfold. 3A moPrP misfolds to a ß-rich dimer in the absence of salt, which can rapidly form an oligomer upon the addition of salt. In the presence of salt, 3A moPrP misfolds to a ß-rich oligomer about a thousand-fold faster than wt moPrP. Importantly, the misfolded structure of the dimer is similar to that of the salt-induced oligomer. Misfolding to oligomer seems to be induced at the level of the dimeric unit by monomer-monomer association, and the oligomer grows by accretion of misfolded dimeric units. Additionally, it is shown that the conserved aromatic residues collectively stabilize not only monomeric protein, but also the structural core of the ß-rich oligomers. Finally, it is also shown that 3A moPrP misfolds much faster to amyloid-fibrils than does the wt protein.

Proliferative arrest induces neuron differentiation and innate immune responses in control and Creutzfeldt-Jakob Disease agent infected rat septal neurons.

Rat post-mitotic septal (SEP) neurons, engineered to conditionally proliferate at 33°C, differentiate when arrested at 37.5°C and can be maintained for weeks without cytotoxic effects. Nine independent cDNA libraries were made to follow arrest-induced neural differentiation and innate immune responses in normal (Nl) uninfected and CJ agent infected SEP cells. Proliferating Nl versus latently infected (CJ-) cells showed few RNA-seq differences. However arrest induced major changes. Normal cells displayed a plethora of anti-proliferative transcripts. Additionally, known neuron differentiation transcripts, e.g., Agtr2, Neuregulin-1, GDF6, SFRP4 and Prnp were upregulated. These Nl neurons also displayed many activated IFN innate immune genes, e.g., OAS1, RTP4, ISG20, GTB4, CD80 and cytokines, complement, and clusterin (CLU) that binds to misfolded proteins. In contrast, arrested highly infectious CJ+ cells (10 logs/gm) downregulated many replication controls. Furthermore, arrested CJ+ cells suppressed neuronal differentiation transcripts, including Prnp which is essential for CJ agent infection. CJ+ cells also enhanced IFN stimulated pathways, and analysis of the 342 CJ+ unique transcripts revealed additional innate immune and anti-viral-linked transcripts, e.g., Il17, ISG15, and RSAD2 (viperin). These data show: 1) innate immune transcripts are produced by normal neurons during differentiation; 2) CJ infection can enhance and expand anti-viral responses; 3) latent CJ infection epigenetically imprints many proliferative pathways to thwart complete arrest. CJ+ brain microglia, white blood cells and intestinal myeloid cells with shared transcripts may be stimulated to educe latent CJD infections that can be clinically silent for >30 years.

Clinical application of whole genome sequencing in young onset dementia: challenges and opportunities.

INTRODUCTION: Young onset dementia (YOD) by its nature is difficult to diagnose. Despite involvement of multidisciplinary neurogenetics services, patients with YOD and their families face significant diagnostic delays. Genetic testing for people with YOD currently involves a staggered, iterative approach. There is currently no optimal single genetic investigation that simultaneously identifies the different genetic variants resulting in YOD. AREAS COVERED: This review discusses the advances in clinical genomic testing for people with YOD. Whole genome sequencing (WGS) can be employed as a 'one stop shop' genomic test for YOD. In addition to single nucleotide variants, WGS can reliably detect structural variants, short tandem repeat expansions, mitochondrial genetic variants as well as capture single nucleotide polymorphisms for the calculation of polygenic risk scores. EXPERT OPINION: WGS, when used as the initial genetic test, can enhance the likelihood of a precision diagnosis and curtail the time taken to reach this. Finding a clinical diagnosis using WGS can reduce invasive and expensive investigations and could be cost effective. These advances need to be balanced against the limitations of the technology and the genetic counseling needs for these vulnerable patients and their families.

Involvement of the nigrostriatal system in Gerstman-Sträussler-Scheinker disease with the PRNP-P102L mutation.

INTRODUCTION: Gerstmann-Sträussler-Scheinker disease (GSS) is an autosomal-dominant inherited prion disease most often associated with the human prion protein gene (PRNP)-P102L mutation. Although patients manifest considerable phenotypic heterogeneity, the involvement of the nigrostriatal system has not been well-studied. METHODS: We performed dopamine transporter single-photon emission computed tomography (DAT-SPECT) using 123I-ioflupane to investigate the nigrostriatal system function in nine patients with the PRNP-P102L mutation. We also examined the pathological findings in another patient whose predominant feature was ataxia and who died 5 years after disease onset. RESULTS: Striatum uptake of 123I-ioflupane indicated by specific binding ratio (SBR) values was significantly reduced in two patients. The DAT-SPECT examination was performed 6 months after disease onset in one of these patients who manifested rapidly developing cognitive decline mimicking Creutzfeldt-Jakob disease. DAT-SPECT was also performed 9 years after disease onset in another patient who manifested the conventional features of GSS involving ataxia and dementia in the initial phase but showed akinetic mutism at the examination time. Another patient examined 2 years after disease onset who predominantly manifested ataxia showed marginally abnormal SBR values. An autopsy case showed moderate neuronal loss in the substantia nigra, and the degree of neuronal loss was similar in most other parts of the brain. CONCLUSION: Nigrostriatal system involvement may occur in patients with GSS associated with the PRNP-P102L mutation, even though parkinsonism is not the predominant feature.

Parkinsonism as an initial presentation of Creutzfeldt-Jakob disease: A case report and review of literature.

Key Clinical Message: Creutzfeldt-Jakob disease (CJD) is a rapidly progressive, fatal neurodegenerative disorder. This case highlights parkinsonism as a rare initial manifestation of sporadic CJD (sCJD), emphasizing the need for heightened clinical awareness to prevent misdiagnosis. Early and accurate diagnosis of sCJD is crucial for preventing potential iatrogenic transmission and optimizing patient management. Abstract: Creutzfeldt-Jakob disease (CJD) is a fatal neurodegenerative illness. While movement disorders may be present at the onset of the disease in about half of those with sporadic CJD (sCJD), parkinsonism is a rare initial presentation. In this article, we report a case of CJD with parkinsonism as the initial presentation of the disease. We report a 69-year-old lady with initial symptoms of gait difficulty, tremor, and bradykinesia. Later, she developed cognitive impairment, ataxia, chin tremor, and myoclonic jerks. Her condition worsened to the point of akinetic mutism. She was diagnosed with probable sCJD after detecting protein 14-3-3 in her cerebrospinal fluid and observing typical imaging features.This case report illustrates important aspects of an inevitably fatal and rapidly progressing disease's early presentation and clinical features. The uncommon initial presentations of sCJD should be considered with the intent of preventing misdiagnosis in the future. Early diagnosis of sCJD can prevent possible iatrogenic disease transmission and improve patient care.

Adaptation of the protein misfolding cyclic amplification (PMCA) technique for the screening of anti-prion compounds.

Prion diseases result from the misfolding of the physiological prion protein (PrPC) to a pathogenic conformation (PrPSc). Compelling evidence indicates that prevention and/or reduction of PrPSc replication are promising therapeutic strategies against prion diseases. However, the existence of different PrPSc conformations (or strains) associated with disease represents a major problem when identifying anti-prion compounds. Efforts to identify strain-specific anti-prion molecules are limited by the lack of biologically relevant high-throughput screening platforms to interrogate compound libraries. Here, we describe adaptations to the protein misfolding cyclic amplification (PMCA) technology (able to faithfully replicate PrPSc strains) that increase its throughput to facilitate the screening of anti-prion molecules. The optimized PMCA platform includes a reduction in sample and reagents, as well as incubation/sonication cycles required to efficiently replicate and detect rodent-adapted and cervid PrPSc strains. The visualization of PMCA products was performed via dot blots, a method that contributed to reduced processing times. These technical changes allowed us to evaluate small molecules with previously reported anti-prion activity. This proof-of-principle screening was evaluated for six rodent-adapted prion strains. Our data show that these compounds targeted either none, all or some PrPSc strains at variable concentrations, demonstrating that this PMCA system is suitable to test compound libraries for putative anti-prion molecules targeting specific PrPSc strains. Further analyses of a small compound library against deer prions demonstrate the potential of this new PMCA format to identify strain-specific anti-prion molecules. The data presented here demonstrate the use of the PMCA technique in the selection of prion strain-specific anti-prion compounds.

RNA-Seq Analysis of Mammalian Prion Disease.

A protein, which can attain a prion state, differs from standard proteins in terms of structural conformation and aggregation propensity. High-throughput sequencing technology provides an opportunity to gain insight into the prion disease condition when coupled with single-cell RNA-Seq analysis to reveal transcriptional changes during prion-based pathogenicity. In this chapter, we present a protocol for RNA-Seq analysis of mammalian prion disease using a single-cell RNA sequencing dataset procured from the NCBI GEO database. This protocol is a tool that can assist researchers in characterizing mammalian prion disease in a reproducible and reusable manner. Further, the resulting output has the potential to provide transcript biomarkers for mammalian prion diseases, which can be employed for diagnostic and prognostic purposes.

Neuropathologically-directed profiling of PRNP somatic and germline variants in sporadic human prion disease.

Creutzfeldt-Jakob Disease (CJD), the most common human prion disease, is associated with pathologic misfolding of the prion protein (PrP), encoded by the PRNP gene. Of human prion disease cases, ~1% were transmitted by misfolded PrP, ~15% are inherited, and ~85% are sporadic (sCJD). While familial cases are inherited through germline mutations in PRNP, the cause of sCJD is unknown. Somatic mutations have been hypothesized as a cause of sCJD, and recent studies have revealed that somatic mutations accumulate in neurons during aging. To investigate the hypothesis that somatic mutations in PRNP may underlie sCJD, we performed deep DNA sequencing of PRNP in 205 sCJD cases and 170 age-matched non-disease controls. We included 5 cases of Heidenhain variant sporadic CJD (H-sCJD), where visual symptomatology and neuropathology implicate focal initiation of prion formation, and examined multiple regions across the brain including in the affected occipital cortex. We employed Multiple Independent Primer PCR Sequencing (MIPP-Seq) with a median depth of >5,000X across the PRNP coding region and analyzed for variants using MosaicHunter. An allele mixing experiment showed positive detection of variants in bulk DNA at a variant allele fraction (VAF) as low as 0.2%. We observed multiple polymorphic germline variants among individuals in our cohort. However, we did not identify bona fide somatic variants in sCJD, including across multiple affected regions in H-sCJD, nor in control individuals. Beyond our stringent variant-identification pipeline, we also analyzed VAFs from raw sequencing data, and observed no evidence of prion disease enrichment for the known germline pathogenic variants P102L, D178N, and E200K. The lack of PRNP pathogenic somatic mutations in H-sCJD or the broader cohort of sCJD suggests that clonal somatic mutations may not play a major role in sporadic prion disease. With H-sCJD representing a focal presentation of neurodegeneration, this serves as a test of the potential role of clonal somatic mutations in genes known to cause familial neurodegeneration.

Genetic insights into drug targets for sporadic Creutzfeldt-Jakob disease: Integrative multi-omics analysis.

OBJECTIVE: Sporadic Creutzfeldt-Jakob disease (sCJD) is a fatal rapidly progressive neurodegenerative disorder with no effective therapeutic interventions. We aimed to identify potential genetically-supported drug targets for sCJD by integrating multi-omics data. METHODS: Multi-omics-wide association studies, Mendelian randomization, and colocalization analyses were employed to explore potential therapeutic targets using expression, single-cell expression, DNA methylation, and protein quantitative trait locus data from blood and brain tissues. Outcome data was from a case-control genome-wide association study, which included 4110 sCJD patients and 13,569 controls. Further investigations encompassed druggability, potential side effects, and associated biological pathways of the identified targets. RESULTS: Integrative multi-omics analysis identified 23 potential therapeutic targets for sCJD, with five targets (STX6, XYLT2, PDIA4, FUCA2, KIAA1614) having higher levels of evidence. One target (XYLT2) shows promise for repurposing, two targets (XYLT2, PDIA4) are druggable, and three (STX6, KIAA1614, and FUCA2) targets represent potential future breakthrough points. The expression level of STX6 and XYLT2 in neurons and oligodendrocytes was closely associated with an increased risk of sCJD. Brain regions with high expression of STX6 or causal links to sCJD were often those areas commonly affected by sCJD. CONCLUSIONS: Our study identified five potential therapeutic targets for sCJD. Further investigations are warranted to elucidate the mechanisms underlying the new targets for developing disease therapies or initiate clinical trials.

Prion diseases, always a threat?

Prion diseases are caused by prions, which are proteinaceous infectious particles that have been identified as causative factors of transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease (CJD). Prion diseases are devastating neurodegenerative disorders in humans and many animals, including sheep, cows, deer, cats, and camels. Prion diseases are classified into sporadic and genetic forms. Additionally, a third, environmentally acquired category exists. This type includes kuru, iatrogenic CJD caused by human dura mater grafts or human pituitary-derived hormones, and variant CJD transmitted through food contaminated with bovine spongiform encephalopathy prions. Bovine spongiform encephalopathy and variant CJD have nearly been controlled, but chronic wasting disease, a prion disease affecting deer, is spreading widely in North America and South Korea and recently in Northern Europe. Recently, amyloid-beta, alpha-synuclein, and other proteins related to Alzheimer's disease, Parkinson's disease, and other neurodegenerative diseases were reported to have prion features such as transmission to animals. Amyloid-beta transmission to humans has been suggested in iatrogenic CJD cases and in cerebral amyloid angiopathy cases with cerebral bleeding occurring long after childhood neurosurgery with or without cadaveric dura mater transplantation. These findings indicate that diseases caused by various prions, namely various transmissible proteins, appear to be a threat, particularly in the current longevity society. Prion disease represented by CJD has obvious transmissibility and is considered to be an "archetype of various neurodegenerative diseases". Overcoming prion diseases is a top priority currently in our society, and this strategy will certainly contribute to elucidating pathomechanism of other neurodegenerative diseases and developing new therapies for them.

18F-FDG PET/CT in early phase of sporadic Creutzfeldt-Jacob disease: A case report.

Key Clinical Message: Creutzfeldt-Jakob disease is a neurodegenerative disorder caused by brain accumulation of a misfolded form of the cellular prion protein, whose diagnosis is challenging, particularly in early stages, due to the variability and nonspecificity of the clinical and radiological features. 18F-fluorodeoxyglucose positron-emitted tomography has the potential to be considered a crucial investigation in these patients, revealing metabolic abnormalities earlier than the conventional neuroimaging analysis. Abstract: A 59-year-old man, the military officer, was referred to our Units for the onset of neurological symptoms rapidly evolving within a month, characterized by akinetic mutism, constructional apraxia, and disorders of spatial orientation. Brain 18F-fluorodeoxyglucose (18F-FDG) positron-emitted tomography (PET)/CT depicted an asymmetric hypometabolism in the left fronto-temporo-parietal cortex, as well as in the left thalamus and the right cerebellar hemisphere, while the glucose metabolism appears to be preserved in the somatosensory cortex and the basal ganglia. Laboratory routine analyses, cerebrospinal fluid routine, infective tests, electroencephalography (EEG), and brain magnetic resonance (MR) were all unremarkable. A positive RT-QuIC result on cerebro-spinal fluid (CSF) was subsequently shown, without any pathogenic gene mutations and, therefore, the result was consistent with a diagnosis of sporadic Creutzfeld-Jacob disease. The clinical evolution was quickly unfavorable, and the patient died about 4 months after hospital admission. FDG PET/computed tomography (CT) has the potential to be considered a crucial investigation in these patients, documenting metabolic changes long time before other diagnostic investigations such as CSF, EEG, brain CT, and brain MR, thus suggesting a greater sensitivity of glucose metabolic evaluation in the early stage of the disease in question.

Genetic characterization of the prion protein gene in camels (Camelus) with comments on the evolutionary history of prion disease in Cetartiodactyla.

Transmissible spongiform encephalopathies (TSEs) are a fatal neurogenerative disease that include Creutzfeldt-Jakob disease in humans, scrapie in sheep and goats, bovine spongiform encephalopathy (BSE), and several others as well as the recently described camel prion disease (CPD). CPD originally was documented in 3.1% of camels examined during an antemortem slaughterhouse inspection in the Ouargla region of Algeria. Of three individuals confirmed for CPD, two were sequenced for the exon 3 of the prion protein gene (PRNP) and were identical to sequences previously reported for Camelus dromedarius. Given that other TSEs, such as BSE, are known to be capable of cross-species transmission and that there is household consumption of meat and milk from Camelus, regulations to ensure camel and human health should be a One Health priority in exporting countries. Although the interspecies transmissibility of CPD currently is unknown, genotypic characterization of Camelus PRNP may be used for predictability of predisposition and potential susceptibility to CPD. Herein, eight breeds of dromedary camels from a previous genetic (mitochondrial DNA and microsatellites) and morphological study were genotyped for PRNP and compared to genotypes from CPD-positive Algerian camels. Sequence data from PRNP indicated that Ethiopian camels possessed 100% sequence identity to CPD-positive camels from Algeria. In addition, the camel PRNP genotype is unique compared to other members of the Orders Cetartiodactyla and Perissodactyla and provides an in-depth phylogenetic analysis of families within Cetartiodactyla and Perissodactyla that was used to infer the evolutionary history of the PRNP gene.

The first report of single nucleotide polymorphisms in the open reading frame of the prion-like protein gene in rabbits.

Background: Natural cases of prion disease have not been reported in rabbits, and prior attempts to identify a prion conversion agent have been unsuccessful. However, recent applications of prion seed amplifying experimental techniques have sparked renewed interest in the potential susceptibility of rabbits to prion disease infections. Among several factors related to prion disease, polymorphisms within the prion-like protein gene (PRND), a member of the prion protein family, have been reported as significantly associated with disease susceptibility in various species. Therefore, our study aimed to investigate polymorphisms in the PRND gene of rabbits and analyze their genetic characteristics. Methods: Genomic DNA was extracted from 207 rabbit samples to investigate leporine PRND polymorphisms. Subsequently, amplicon sequencing targeting the coding region of the leporine PRND gene was conducted. Additionally, linkage disequilibrium (LD) analysis was employed to assess the connection within and between loci. The impact of non-synonymous single nucleotide polymorphisms (SNPs) on the Doppel protein was evaluated using PolyPhen-2. Results: We found nine novel SNPs in the leporine PRND gene: c.18A > G, c.76G > C, c.128C > T, c.146C > T, c.315A > G, c.488G > A, c.525G > C, c.544G > A, and c.579A > G. Notably, seven of these PRND SNPs, excluding c.525G > C and c.579A > G, exhibited strong LD values exceeding 0.3. In addition, LD analysis confirmed a robust link between PRNP SNP c.234C > T and PRND SNPs at c.525G > C and c.579A > G. Furthermore, according to PolyPhen-2 and SIFT analyses, the four non-synonymous SNPs were predicted to have deleterious effects on the function or structure of the Doppel protein. However, PANTHER and Missense3D did not indicate such effects. Conclusion: In this paper, we have identified novel SNPs in the rabbit PRND gene and predicted their potential detrimental effects on protein function or structure through four non-synonymous SNPs. Additionally, we observed a genetic linkage between SNPs in the PRND and PRNP genes. These findings may provide insights into understanding the characteristics of rabbits as partially resistant species. To the best of our knowledge, this study is the first to genetically characterize PRND SNPs in rabbits.

Rational Tuning of the Concentration-independent Enrichment of Prion-like Domains in Stress Granules.

Stress granules (SGs) are large ribonucleoprotein assemblies that form in response to acute stress in eukaryotes. SG formation is thought to be initiated by liquid-liquid phase separation (LLPS) of key proteins and RNA. These molecules serve as a scaffold for recruitment of client molecules. LLPS of scaffold proteins in vitro is highly concentration-dependent, yet biomolecular condensates in vivo contain hundreds of unique proteins, most of which are thought to be clients rather than scaffolds. Many proteins that localize to SGs contain low-complexity, prion-like domains (PrLDs) that have been implicated in LLPS and SG recruitment. The degree of enrichment of proteins in biomolecular condensates such as SGs can vary widely, but the underlying basis for these differences is not fully understood. Here, we develop a toolkit of model PrLDs to examine the factors that govern efficiency of PrLD recruitment to stress granules. Recruitment was highly sensitive to amino acid composition: enrichment in SGs could be tuned through subtle changes in hydrophobicity. By contrast, SG recruitment was largely insensitive to PrLD concentration at both a population level and single-cell level. These observations point to a model wherein PrLDs are enriched in SGs through either simple solvation effects or interactions that are effectively non-saturable even at high expression levels.

Lack of cellular prion protein causes Amyloid ß accumulation, increased extracellular vesicle abundance, and changes to exosome biogenesis proteins.

Alzheimer's disease (AD) progression is closely linked to the propagation of pathological Amyloid ß (Aß), a process increasingly understood to involve extracellular vesicles (EVs), namely exosomes. The specifics of Aß packaging into exosomes remain elusive, although evidence suggests an ESCRT (Endosomal Sorting Complex Required for Transport)-independent origin to be responsible in spreading of AD pathogenesis. Intriguingly, PrPC, known to influence exosome abundance and bind oligomeric Aß (oAß), can be released in exosomes via both ESCRT-dependent and ESCRT-independent pathways, raising questions about its role in oAß trafficking. Thus, we quantified Aß levels within EVs, cell medium, and intracellularly, alongside exosome biogenesis-related proteins, following deletion or overexpression of PrPC. The same parameters were also evaluated in the presence of specific exosome inhibitors, namely Manumycin A and GW4869. Our results revealed that deletion of PrPC increases intracellular Aß accumulation and amplifies EV abundance, alongside significant changes in cellular levels of exosome biogenesis-related proteins Vps25, Chmp2a, and Rab31. In contrast, cellular expression of PrPC did not alter exosomal Aß levels. This highlights PrPC's influence on exosome biogenesis, albeit not in direct Aß packaging. Additionally, our data confirm the ESCRT-independent exosome release of Aß and we show a direct reduction in Chmp2a levels upon oAß challenge. Furthermore, inhibition of opposite exosome biogenesis pathway resulted in opposite cellular PrPC levels. In conclusion, our findings highlight the intricate relationship between PrPC, exosome biogenesis, and Aß release. Specifically, they underscore PrPC's critical role in modulating exosome-associated proteins, EV abundance, and cellular Aß levels, thereby reinforcing its involvement in AD pathogenesis.