Anti-prion drugs do not improve survival in novel knock-in models of inherited prion disease.

Prion diseases uniquely manifest in three distinct forms: inherited, sporadic, and infectious. Wild-type prions are responsible for the sporadic and infectious versions, while mutant prions cause inherited variants like fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD). Although some drugs can prolong prion incubation times up to four-fold in rodent models of infectious prion diseases, no effective treatments for FFI and fCJD have been found. In this study, we evaluated the efficacy of various anti-prion drugs on newly-developed knock-in mouse models for FFI and fCJD. These models express bank vole prion protein (PrP) with the pathogenic D178N and E200K mutations. We applied various drug regimens known to be highly effective against wild-type prions in vivo as well as a brain-penetrant compound that inhibits mutant PrPSc propagation in vitro. None of the regimens tested (Anle138b, IND24, Anle138b + IND24, cellulose ether, and PSCMA) significantly extended disease-free survival or prevented mutant PrPSc accumulation in either knock-in mouse model, despite their ability to induce strain adaptation of mutant prions. Our results show that anti-prion drugs originally developed to treat infectious prion diseases do not necessarily work for inherited prion diseases, and that the recombinant sPMCA is not a reliable platform for identifying compounds that target mutant prions. This work underscores the need to develop therapies and validate screening assays specifically for mutant prions, as well as anti-prion strategies that are not strain-dependent.

Wnt, glucocorticoid and cellular prion protein cooperate to drive a mesenchymal phenotype with poor prognosis in colon cancer.

BACKGROUND: The mesenchymal subtype of colorectal cancer (CRC), associated with poor prognosis, is characterized by abundant expression of the cellular prion protein PrPC, which represents a candidate therapeutic target. How PrPC is induced in CRC remains elusive. This study aims to elucidate the signaling pathways governing PrPC expression and to shed light on the gene regulatory networks linked to PrPC. METHODS: We performed in silico analyses on diverse datasets of in vitro, ex vivo and in vivo models of mouse CRC and patient cohorts. We mined ChIPseq studies and performed promoter analysis. CRC cell lines were manipulated through genetic and pharmacological approaches. We created mice combining conditional inactivation of Apc in intestinal epithelial cells and overexpression of the human prion protein gene PRNP. Bio-informatic analyses were carried out in two randomized control trials totalizing over 3000 CRC patients. RESULTS: In silico analyses combined with cell-based assays identified the Wnt-ß-catenin and glucocorticoid pathways as upstream regulators of PRNP expression, with subtle differences between mouse and human. We uncover multiple feedback loops between PrPC and these two pathways, which translate into an aggravation of CRC pathogenesis in mouse. In stage III CRC patients, the signature defined by PRNP-CTNNB1-NR3C1, encoding PrPC, ß-catenin and the glucocorticoid receptor respectively, is overrepresented in the poor-prognosis, mesenchymal subtype and associates with reduced time to recurrence. CONCLUSIONS: An unleashed PrPC-dependent vicious circle is pathognomonic of poor prognosis, mesenchymal CRC. Patients from this aggressive subtype of CRC may benefit from therapies targeting the PRNP-CTNNB1-NR3C1 axis.

Sensitive detection of pathological seeds of α-synuclein, tau and prion protein on solid surfaces.

Prions or prion-like aggregates such as those composed of PrP, α-synuclein, and tau are key features of proteinopathies such as prion, Parkinson's and Alzheimer's diseases, respectively. Their presence on solid surfaces may be biohazardous under some circumstances. PrP prions bound to solids are detectable by ultrasensitive real-time quaking-induced conversion (RT-QuIC) assays if the solids can be immersed in assay wells or the prions transferred to pads. Here we show that prion-like seeds can remain detectable on steel wires for at least a year, or even after enzymatic cleaning and sterilization. We also show that contamination of larger objects with pathological seeds of α-synuclein, tau, and PrP can be detected by simply assaying a sampling medium that has been transiently applied to the surface. Human α-synuclein seeds in dementia with Lewy bodies brain tissue were detected by α-synuclein RT-QuIC after drying of tissue dilutions with concentrations as low as 10-6 onto stainless steel. Tau RT-QuIC detected tau seeding activity on steel exposed to Alzheimer's disease brain tissue diluted as much as a billion fold. Prion RT-QuIC assays detected seeding activity on plates exposed to brain dilutions as extreme as 10-5-10-8 from prion-affected humans, sheep, cattle and cervids. Sampling medium collected from surgical instruments used in necropsies of sporadic Creutzfeldt-Jakob disease-infected transgenic mice was positive down to 10-6 dilution. Sensitivity for prion detection was not sacrificed by omitting the recombinant PrP substrate from the sampling medium during its application to a surface and subsequent storage as long as the substrate was added prior to performing the assay reaction. Our findings demonstrate practical prototypic surface RT-QuIC protocols for the highly sensitive detection of pathologic seeds of α-synuclein, tau, and PrP on solid objects.

miR-519a-3p, found to regulate cellular prion protein during Alzheimer's disease pathogenesis, as a biomarker of asymptomatic stages.

Clinical relevance of miRNAs as biomarkers is growing due to their stability and detection in biofluids. In this, diagnosis at asymptomatic stages of Alzheimer's disease (AD) remains a challenge since it can only be made at autopsy according to Braak NFT staging. Achieving the objective of detecting AD at early stages would allow possible therapies to be addressed before the onset of cognitive impairment. Many studies have determined that the expression pattern of some miRNAs is dysregulated in AD patients, but to date, none has been correlated with downregulated expression of cellular prion protein (PrPC) during disease progression. That is why, by means of cross studies of miRNAs up-regulated in AD with in silico identification of potential miRNAs-binding to 3'UTR of human PRNP gene, we selected miR-519a-3p for our study. Then, in vitro experiments were carried out in two ways. First, we validated miR-519a-3p target on 3'UTR-PRNP, and second, we analyzed the levels of PrPC expression after using of mimic technology on cell culture. In addition, RT-qPCR was performed to analyzed miR-519a-3p expression in human cerebral samples of AD at different stages of disease evolution. Additionally, samples of other neurodegenerative diseases such as other non-AD tauopathies and several synucleinopathies were included in the study. Our results showed that miR-519a-3p overlaps with PRNP 3'UTR in vitro and promotes downregulation of PrPC. Moreover, miR-519a-3p was found to be up-regulated exclusively in AD samples from stage I to VI, suggesting its potential use as a novel label of preclinical stages of the disease.

VCP/p97 mediates nuclear targeting of non-ER-imported prion protein to maintain proteostasis.

Mistargeting of secretory proteins in the cytosol can trigger their aggregation and subsequent proteostasis decline. We have identified a VCP/p97-dependent pathway that directs non-ER-imported prion protein (PrP) into the nucleus to prevent the formation of toxic aggregates in the cytosol. Upon impaired translocation into the ER, PrP interacts with VCP/p97, which facilitates nuclear import mediated by importin-ß. Notably, the cytosolic interaction of PrP with VCP/p97 and its nuclear import are independent of ubiquitination. In vitro experiments revealed that VCP/p97 binds non-ubiquitinated PrP and prevents its aggregation. Inhibiting binding of PrP to VCP/p97, or transient proteotoxic stress, promotes the formation of self-perpetuating and partially proteinase resistant PrP aggregates in the cytosol, which compromised cellular proteostasis and disrupted further nuclear targeting of PrP. In the nucleus, RNAs keep PrP in a soluble and non-toxic conformation. Our study revealed a novel ubiquitin-independent role of VCP/p97 in the nuclear targeting of non-imported secretory proteins and highlights the impact of the chemical milieu in triggering protein misfolding.

Generation of an induced pluripotent stem cell line (PNUYHi002-A) from a patient with Alzheimer's disease carrying PRNP M232R variant.

We generated a human induced pluripotent stem cell (iPSC) line from the peripheral blood mononuclear cells isolated from a 59-year-old male patient with Alzheimer's disease (AD). The iPSC line was meticulously characterized to confirm its pluripotency, absence of transgenes, and normal karyotype. The unexpected discovery of the M232R variant in PRNP makes this cell line a valuable resource for investigating AD pathogenesis.

Variably protease-sensitive prionopathy with methionine homozygosity at codon 129 in the prion protein gene.

Variably protease-sensitive prionopathy (VPSPr) is a recently characterised rare subtype of sporadic prion disease, mainly affecting individuals with valine homozygosity at codon 129 in the prion protein gene, with only seven methionine homozygote cases reported to date. This case presents clinical, neuropathological and biochemical features of the eighth VPSPr case worldwide with methionine homozygosity at codon 129 and compares the features with the formerly presented cases.The patient, a woman in her 70s, presented with cognitive decline, impaired balance and frequent falls. Medical history and clinical presentation were suggestive of a rapidly progressive dementia disorder. MRI showed bilateral thalamic hyperintensity. Cerebrospinal fluid real-time quaking-induced conversion was negative, and the electroencephalogram was unremarkable. The diagnosis was established through post-mortem pathological examinations. VPSPr should be suspected in rapidly progressive dementia lacking typical features or paraclinical results of protein misfolding diseases.

Creutzfeldt-Jakob disease and other prion diseases.

Prion diseases share common clinical and pathological characteristics such as spongiform neuronal degeneration and deposition of an abnormal form of a host-derived protein, termed prion protein. The characteristic features of prion diseases are long incubation times, short clinical courses, extreme resistance of the transmissible agent to degradation and lack of nucleic acid involvement. Sporadic and genetic forms of prion diseases occur worldwide, of which genetic forms are associated with mutations in PRNP. Human to human transmission of these diseases has occurred due to iatrogenic exposure, and zoonotic forms of prion diseases are linked to bovine disease. Significant progress has been made in the diagnosis of these disorders. Clinical tools for diagnosis comprise brain imaging and cerebrospinal fluid tests. Aggregation assays for detection of the abnormally folded prion protein have a clear potential to diagnose the disease in peripherally accessible biofluids. After decades of therapeutic nihilism, new treatment strategies and clinical trials are on the horizon. Although prion diseases are relatively rare disorders, understanding their pathogenesis and mechanisms of prion protein misfolding has significantly enhanced the field in research of neurodegenerative diseases.

Integrated transcriptomics uncovers an enhanced association between the prion protein gene expression and vesicle dynamics signatures in glioblastomas.

BACKGROUND: Glioblastoma (GBM) is an aggressive brain tumor that exhibits resistance to current treatment, making the identification of novel therapeutic targets essential. In this context, cellular prion protein (PrPC) stands out as a potential candidate for new therapies. Encoded by the PRNP gene, PrPC can present increased expression levels in GBM, impacting cell proliferation, growth, migration, invasion and stemness. Nevertheless, the exact molecular mechanisms through which PRNP/PrPC modulates key aspects of GBM biology remain elusive. METHODS: To elucidate the implications of PRNP/PrPC in the biology of this cancer, we analyzed publicly available RNA sequencing (RNA-seq) data of patient-derived GBMs from four independent studies. First, we ranked samples profiled by bulk RNA-seq as PRNPhigh and PRNPlow and compared their transcriptomic landscape. Then, we analyzed PRNP+ and PRNP- GBM cells profiled by single-cell RNA-seq to further understand the molecular context within which PRNP/PrPC might function in this tumor. We explored an additional proteomics dataset, applying similar comparative approaches, to corroborate our findings. RESULTS: Functional profiling revealed that vesicular dynamics signatures are strongly correlated with PRNP/PrPC levels in GBM. We found a panel of 73 genes, enriched in vesicle-related pathways, whose expression levels are increased in PRNPhigh/PRNP+ cells across all RNA-seq datasets. Vesicle-associated genes, ANXA1, RAB31, DSTN and SYPL1, were found to be upregulated in vitro in an in-house collection of patient-derived GBM. Moreover, proteome analysis of patient-derived samples reinforces the findings of enhanced vesicle biogenesis, processing and trafficking in PRNPhigh/PRNP+ GBM cells. CONCLUSIONS: Together, our findings shed light on a novel role for PrPC as a potential modulator of vesicle biology in GBM, which is pivotal for intercellular communication and cancer maintenance. We also introduce GBMdiscovery, a novel user-friendly tool that allows the investigation of specific genes in GBM biology.

Ecology and Chronic Wasting Disease Epidemiology Shape Prion Protein Gene Variation in Rocky Mountain Elk (Cervus elaphus nelsoni).

As chronic wasting disease (CWD) continues to spread across North America, the relationship between CWD and host genetics has become of interest. In Rocky Mountain elk (Cervus elaphus nelsoni), one or two copies of a leucine allele at codon 132 of the prion protein gene (132L*) has been shown to prolong the incubation period of CWD. Our study examined the relationship between CWD epidemiology and codon 132 evolution in elk from Wyoming, USA, from 2011 to 2018. Using PCR and Sanger sequencing, we genotyped 997 elk and assessed the relationship between genotype and CWD prevalence estimated from surveillance data. Using logistic regression, we showed that each 1% increase in CWD prevalence is associated with a 9.6% increase in the odds that an elk would have at least one copy of leucine at codon 132. In some regions, however, 132L* variants were found in the absence of CWD, indicating that evolutionary and epidemiologic patterns can be heterogeneous across space and time. We also provide evidence that naturally occurring CWD is not rare in 132L* elk, which merits the study of shedding kinetics in 132L* elk and the influence of genotype on CWD strain diversity. The management implications of cervid adaptations to CWD are difficult to predict. Studies that investigate the degree to which evolutionary outcomes are shaped by host spatial structure can provide useful epidemiologic insight, which can in turn aid management by informing scale and extent of mitigation actions.

Efficient transmission of human prion diseases to a glycan-free prion protein-expressing host.

It is increasingly evident that the association of glycans with the prion protein (PrP), a major post-translational modification, significantly impacts the pathogenesis of prion diseases. A recent bioassay study has provided evidence that the presence of PrP glycans decreases spongiform degeneration and disease-related PrP (PrPD) deposition in a murine model. We challenged (PRNPN181Q/197Q) transgenic (Tg) mice expressing glycan-free human PrP (TgGlyc-), with isolates from sporadic Creutzfeldt-Jakob disease subtype MM2 (sCJDMM2), sporadic fatal insomnia and familial fatal insomnia, three human prion diseases that are distinct but share histotypic and PrPD features. TgGlyc- mice accurately replicated the basic histotypic features associated with the three diseases but the transmission was characterized by high attack rates, shortened incubation periods and a greatly increased severity of the histopathology, including the presence of up to 40 times higher quantities of PrPD that formed prominent deposits. Although the engineered protease-resistant PrPD shared at least some features of the secondary structure and the presence of the anchorless PrPD variant with the wild-type PrPD, it exhibited different density gradient profiles of the PrPD aggregates and a higher stability index. The severity of the histopathological features including PrP deposition appeared to be related to the incubation period duration. These findings are clearly consistent with the protective role of the PrP glycans but also emphasize the complexity of the conformational changes that impact PrPD following glycan knockout. Future studies will determine whether these features apply broadly to other human prion diseases or are PrPD-type dependent.

Inhibition of the RBMS1/PRNP axis improves ferroptosis resistance-mediated oxaliplatin chemoresistance in colorectal cancer.

The majority of patients with advanced colorectal cancer have chemoresistance to oxaliplatin, and studies on oxaliplatin resistance are limited. Our research showed that RNA-binding motif single-stranded interacting protein 1 (RBMS1) caused ferroptosis resistance in tumor cells, leading to oxaliplatin resistance. We employed bioinformatics to evaluate publically accessible data sets and discovered that RBMS1 was significantly upregulated in oxaliplatin-resistant colorectal cancer cells, in tandem with ferroptosis suppression. In vivo and in vitro studies revealed that inhibiting RBMS1 expression caused ferroptosis in colorectal cancer cells, restoring tumor cell sensitivity to oxaliplatin. Mechanistically, this is due to RBMS1 inducing prion protein translation, resulting in ferroptosis resistance in tumor cells. Validation of clinical specimens revealed that RBMS1 is similarly linked to tumor development and a poor prognosis. Overall, RBMS1 is a potential therapeutic target with clinical translational potential, particularly for oxaliplatin chemoresistance in colorectal cancer.

Prion Protein Overexpression in Adipose-Derived Mesenchymal Stem Cells (ADMSCs) Effectively Protected Rodent Kidney Against Ischemia-Reperfusion Injury Via Enhancing ATP/Mitochondrial Biogenesis-Role of ADMSC Rejuvenation and Proliferation.

BACKGROUND: We tested the hypothesis that overexpression of cellular-prion-protein in adipose-derived mesenchymal stem cells (PrPCOE-ADMSCs) effectively protected the kidney against ischemia-reperfusion (IR) injury in rat. METHODS: Part I of cell culture was categorized into A1(ADMSCs)/A2(ADMSCs+p-Cresol)/A3(PrPCOE in ADMSCs)/A4 (PrPCOE in ADMSCs+p-Cresol). Part II of cell culture was divided into B1(ADMSCs)/B2[ADMSCs+lipopolysaccharide (LPS)]/B3(PrPCOE in ADMSCs)/B4(PrPCOE in ADMSCs+LPS). Sprague-Dawley (SD) rats (n = 50) were equally categorized into groups 1 (sham-operated-control)/2 (IR)/3 (IR+ADMSCs/6.0 × 105 equally divided into bilateral-renal arteries and 6.0 × 105 intravenous administration by 1 h after IR)/4 [IR+PrPCOE-ADMSCs (identical dosage administered as group 3)]/5 [IR+silencing PRNP -ADMSCs (identical dosage administered as group 3)], and kidneys were harvested post-day 3 IR injury. RESULTS: Part I results demonstrated that the cell viability at 24/48/72 h, BrdU uptake/number of mitDNA/APT concentration/mitochondrial-cytochrome-C+ cells and the protein expressions of ki67/PrPC at 72 h-cell culturing were significantly higher in PrPCOE-ADMSCs than in ADMSCs (all P < 0.001). The protein expressions of oxidative-stress (NOX-1/NOX2/NOX4/oxidized protein)/mitochondrial-damaged (p22-phox/cytosolic-cytochrome-C)/inflammatory (p-NF-κB/IL-1ß/TNF-α/IL-6)/apoptotic (cleaved caspase-3/cleaved-PARP) biomarkers were lowest in A1/A3 and significantly higher in A2 than in A4 (all P < 0.001). Part II result showed that the protein expressions of inflammatory (p-NF-κB/IL-1ß/TNF-α/IL-6)/apoptotic (cleaved caspase-3/cleaved-PARP) biomarkers exhibited an identical pattern of part I among the groups (all P < 0.001). The protein expressions of inflammatory (p-NF-κB/IL-1ß/TNF-α/MMP-9)/oxidative-stress (NOX-1/NOX-2/oxidized-protein)/mitochondrial-damaged (cytosolic-cytochrome-C/p22-phox)/apoptotic (cleaved caspase-3/cleaved-PARP/mitochondrial-Bx)/autophagic (beclin-1/ratio of LC3B-II/LC3B-I)/fibrotic (Smad3/TGF-ß) biomarkers and kidney-injury-score/creatinine level were lowest in group 1, highest in group 2, significantly higher in group 5 than in groups 3/4 (all P < 0.0001). CONCLUSION: PrPCOE in ADMSCs rejuvenated these cells and played a cardinal role on protecting the kidney against IR injury.

Mutations of evolutionarily conserved aromatic residues suggest that misfolding of the mouse prion protein may commence in multiple ways.

The misfolding of the mammalian prion protein from its α-helix rich cellular isoform to its ß-sheet rich infectious isoform is associated with several neurodegenerative diseases. The determination of the structural mechanism by which misfolding commences, still remains an unsolved problem. In the current study, native-state hydrogen exchange coupled with mass spectrometry has revealed that the N state of the mouse prion protein (moPrP) at pH 4 is in dynamic equilibrium with multiple partially unfolded forms (PUFs) capable of initiating misfolding. Mutation of three evolutionarily conserved aromatic residues, Tyr168, Phe174, and Tyr217 present at the interface of the ß2-α2 loop and the C-terminal end of α3 in the structured C-terminal domain of moPrP significantly destabilize the native state (N) of the protein. They also reduce the free energy differences between the N state and two PUFs identified as PUF1 and PUF2**. It is shown that PUF2** in which the ß2-α2 loop and the C-terminal end of α3 are disordered, has the same stability as the previously identified PUF2*, but to have a very different structure. Misfolding can commence from both PUF1 and PUF2**, as it can from PUF2*. Hence, misfolding can commence and proceed in multiple ways from structurally distinct precursor conformations. The increased extents to which PUF1 and PUF2** are populated at equilibrium in the case of the mutant variants, greatly accelerate their misfolding. The results suggest that the three aromatic residues may have been evolutionarily selected to impede the misfolding of moPrP.

The yeast prion protein Sup35 initiates α-synuclein pathology in mouse models of Parkinson's disease.

Parkinson's disease (PD) is characterized by the pathologic aggregation and prion-like propagation of α-synuclein (α-syn). Emerging evidence shows that fungal infections increase the incidence of PD. However, the molecular mechanisms by which fungi promote the onset of PD are poorly understood. Here, we show that nasal infection with Saccharomyces cerevisiae (S. cerevisiae) in α-syn A53T transgenic mice accelerates the aggregation of α-syn. Furthermore, we found that Sup35, a prion protein from S. cerevisiae, is the key factor initiating α-syn pathology induced by S. cerevisiae. Sup35 interacts with α-syn and accelerates its aggregation in vitro. Notably, injection of Sup35 fibrils into the striatum of wild-type mice led to α-syn pathology and PD-like motor impairment. The Sup35-seeded α-syn fibrils showed enhanced seeding activity and neurotoxicity compared with pure α-syn fibrils in vitro and in vivo. Together, these observations indicate that the yeast prion protein Sup35 initiates α-syn pathology in PD.

Development of a sensitive real-time quaking-induced conversion (RT-QuIC) assay for application in prion-infected blood.

Efforts to prevent human-to-human transmission of variant Creutzfeldt-Jakob disease (vCJD) by contaminated blood would be aided by the development of a sensitive diagnostic test that could be routinely used to screen blood donations. As blood samples from vCJD patients are extremely rare, here we describe the optimisation of real-time quaking-induced conversion (RT-QuIC) for detection of PrPSc (misfolded prion protein, a marker of prion infection) in blood samples from an established large animal model of vCJD, sheep experimentally infected with bovine spongiform encephalopathy (BSE). Comparative endpoint titration experiments with RT-QuIC, miniaturized bead protein misfolding cyclic amplification (mb-PMCA) and intracerebral inoculation of a transgenic mouse line expressing sheep PrP (tgOvARQ), demonstrated highly sensitive detection of PrPSc by RT-QuIC in a reference sheep brain homogenate. Upon addition of a capture step with iron oxide beads, the RT-QuIC assay was able to detect PrPSc in whole blood samples from BSE-infected sheep up to two years before disease onset. Both RT-QuIC and mb-PMCA also demonstrated sensitive detection of PrPSc in a reference vCJD-infected human brain homogenate, suggesting that either assay may be suitable for application to human blood samples. Our results support the further development and evaluation of RT-QuIC as a diagnostic or screening test for vCJD.

The role of cellular prion protein in immune system.

Numerous studies have investigated the cellular prion protein (PrPC) since its discovery. These investigations have explained that its structure is predominantly composed of alpha helices and short beta sheet segments, and when its abnormal scrapie isoform (PrPSc) is infected, PrPSc transforms the PrPC, leading to prion diseases, including Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cattle. Given its ubiquitous distribution across a variety of cellular types, the PrPC manifests a diverse range of biological functions, including cell-cell adhesion, neuroprotection, signalings, and oxidative stress response. PrPC is also expressed in immune tissues, and its functions in these tissues include the activation of immune cells and the formation of secondary lymphoid tissues, such as the spleen and lymph nodes. Moreover, high expression of PrPC in immune cells plays a crucial role in the pathogenesis of prion diseases. In addition, it affects inflammation and the development and progression of cancer via various mechanisms. In this review, we discuss the studies on the role of PrPC from various immunological perspectives. [BMB Reports 2023; 56(12): 645-650].

Cellular Prion protein moonlights vascular smooth muscle cell fate: Surveilled by trophoblast cells.

Uterine spiral artery remodeling (uSAR) is a hallmark of hemochorial placentation. Compromised uSAR leads to adverse pregnancy outcomes. Salient developmental events involved in uSAR are active areas of research and include (a) trophendothelial cell invasion into the spiral arteries, selected demise of endothelial cells; (b) de-differentiation of vascular smooth muscle cells (VSMC); and (c) migration and/or death of VSMCs surrounding spiral arteries. Here we demonstrated that cellular prion (PRNP) is expressed in the rat metrial gland, the entry point of spiral arteries with the highest expression on E16.5, the day at which trophoblast invasion peaks. PRNP is expressed in VSMCs that drift away from the arterial wall. RNA interference of Prnp functionally restricted migration and invasion of rat VSMCs. Furthermore, PRNP interacted with two migration-promoting factors, focal adhesion kinase (FAK) and platelet-derived growth factor receptor-ß (PDGFR-ß), forming a ter-molecular complex in both the metrial gland and A7r5 cells. The presence of multiple putative binding site of odd skipped related-1 (OSR1) transcription factor on the Prnp promoter was observed using in silico promoter analysis. Ectopic overexpression of OSR1 increased, and knockdown of OSR1 decreased expression of PRNP in VSMCs. Coculture of VSMCs with rat primary trophoblast cells decreased the levels of OSR1 and PRNP. Interestingly, PRNP knockdown led to apoptotic death in ~9% of VSMCs and activated extrinsic apoptotic pathways. PRNP interacts with TRAIL-receptor DR4 and protects VSMCs from TRAIL-mediated apoptosis. These results highlight the biological functions of PRNP in VSMC cell-fate determination during uteroplacental development, an important determinant of healthy pregnancy outcome.

A PrP EGFR signaling axis controls neural stem cell senescence through modulating cellular energy pathways.

Mis-folding of the prion protein (PrP) is known to cause neurodegenerative disease; however, the native function of this protein remains poorly defined. PrP has been linked with many cellular functions, including cellular proliferation and senescence. It is also known to influence epidermal growth factor receptor (EGFR) signaling, a pathway that is itself linked with both cell growth and senescence. Adult neural stem cells (NSCs) persist at low levels in the brain throughout life and retain the ability to proliferate and differentiate into new neural lineage cells. KO of PrP has previously been shown to reduce NSC proliferative capacity. We used PrP KO and WT NSCs from adult mouse brain to examine the influence of PrP on cellular senescence, EGFR signaling, and the downstream cellular processes. PrP KO NSCs showed decreased cell proliferation and increased senescence in in vitro cultures. Expression of EGFR was decreased in PrP KO NSCs compared with WT NSCs and additional supplementation of EGF was sufficient to reduce senescence. RNA-seq analysis confirmed that significant changes were occurring at the mRNA level within the EGFR signaling pathway and these were associated with reduced expression of mitochondrial components and correspondingly reduced mitochondrial function. Metabolomic analysis of cellular energy pathways showed that blockages were occurring at critical sites for production of energy and biomass, including catabolism of pyruvate. We conclude that, in the absence of PrP, NSC growth pathways are downregulated as a consequence of insufficient energy and growth intermediates.

Quantitative analysis of prion disease using an AI-powered digital pathology framework.

Prion disease is a fatal neurodegenerative disorder characterized by accumulation of an abnormal prion protein (PrPSc) in the central nervous system. To identify PrPSc aggregates for diagnostic purposes, pathologists use immunohistochemical staining of prion protein antibodies on tissue samples. With digital pathology, artificial intelligence can now analyze stained slides. In this study, we developed an automated pipeline for the identification of PrPSc aggregates in tissue samples from the cerebellar and occipital cortex. To the best of our knowledge, this is the first framework to evaluate PrPSc deposition in digital images. We used two strategies: a deep learning segmentation approach using a vision transformer, and a machine learning classification approach with traditional classifiers. Our method was developed and tested on 64 whole slide images from 41 patients definitively diagnosed with prion disease. The results of our study demonstrated that our proposed framework can accurately classify WSIs from a blind test set. Moreover, it can quantify PrPSc distribution and localization throughout the brain. This could potentially be extended to evaluate protein expression in other neurodegenerative diseases like Alzheimer's and Parkinson's. Overall, our pipeline highlights the potential of AI-assisted pathology to provide valuable insights, leading to improved diagnostic accuracy and efficiency.