The cellular prion protein does not affect tau seeding and spreading of sarkosyl-insoluble fractions from Alzheimer's disease.

The cellular prion protein (PrPC) plays many roles in the developing and adult brain. In addition, PrPC binds to several amyloids in oligomeric and prefibrillar forms and may act as a putative receptor of abnormal misfolded protein species. The role of PrPC in tau seeding and spreading is not known. In the present study, we have inoculated well-characterized sarkosyl-insoluble fractions of sporadic Alzheimer's disease (sAD) into the brain of adult wild-type mice (Prnp+/+), Prnp0/0 (ZH3 strain) mice, and mice over-expressing the secreted form of PrPC lacking their GPI anchor (Tg44 strain). Phospho-tau (ptau) seeding and spreading involving neurons and oligodendrocytes were observed three and six months after inoculation. 3Rtau and 4Rtau deposits from the host tau, as revealed by inoculating Mapt0/0 mice and by using specific anti-mouse and anti-human tau antibodies suggest modulation of exon 10 splicing of the host mouse Mapt gene elicited by exogenous sAD-tau. However, no tau seeding and spreading differences were observed among Prnp genotypes. Our results show that PrPC does not affect tau seeding and spreading in vivo.

α-Synuclein strain propagation is independent of cellular prion protein expression in a transgenic synucleinopathy mouse model.

The cellular prion protein, PrPC, has been postulated to function as a receptor for α-synuclein, potentially facilitating cell-to-cell spreading and/or toxicity of α-synuclein aggregates in neurodegenerative disorders such as Parkinson's disease. Previously, we generated the "Salt (S)" and "No Salt (NS)" strains of α-synuclein aggregates that cause distinct pathological phenotypes in M83 transgenic mice overexpressing A53T-mutant human α-synuclein. To test the hypothesis that PrPC facilitates the propagation of α-synuclein aggregates, we produced M83 mice that either express or do not express PrPC. Following intracerebral inoculation with the S or NS strain, the absence of PrPC in M83 mice did not prevent disease development and had minimal influence on α-synuclein strain-specified attributes such as the extent of cerebral α-synuclein deposition, selective targeting of specific brain regions and cell types, the morphology of induced α-synuclein deposits, and the structural fingerprints of protease-resistant α-synuclein aggregates. Likewise, there were no appreciable differences in disease manifestation between PrPC-expressing and PrPC-lacking M83 mice following intraperitoneal inoculation of the S strain. Interestingly, intraperitoneal inoculation with the NS strain resulted in two distinct disease phenotypes, indicative of α-synuclein strain evolution, but this was also independent of PrPC expression. Overall, these results suggest that PrPC plays at most a minor role in the propagation, neuroinvasion, and evolution of α-synuclein strains in mice that express A53T-mutant human α-synuclein. Thus, other putative receptors or cell-to-cell propagation mechanisms may have a larger effect on the spread of α-synuclein aggregates during disease.

PrPC controls epithelial-to-mesenchymal transition in EGFR-mutated NSCLC: implications for TKI resistance and patient follow-up.

Patients with EGFR-mutated non-small cell lung cancer (NSCLC) benefit from treatment with tyrosine kinase inhibitors (TKI) targeting EGFR. Despite improvements in patient care, especially with the 3rd generation TKI osimertinib, disease relapse is observed in all patients. Among the various processes involved in TKI resistance, epithelial-to-mesenchymal transition (EMT) is far from being fully characterized. We hypothesized that the cellular prion protein PrPC could be involved in EMT and EGFR-TKI resistance in NSCLC. Using 5 independent lung adenocarcinoma datasets, including our own cohort, we document that the expression of the PRNP gene encoding PrPC is associated with EMT. By manipulating the levels of PrPC in different EGFR-mutated NSCLC cell lines, we firmly establish that the expression of PrPC is mandatory for cells to maintain or acquire a mesenchymal phenotype. Mechanistically, we show that PrPC operates through an ILK-RBPJ cascade, which also controls the expression of EGFR. Our data further demonstrate that PrPC levels are elevated in EGFR-mutated versus wild-type tumours or upon EGFR activation in vitro. In addition, we provide evidence that PRNP levels increase with TKI resistance and that reducing PRNP expression sensitizes cells to osimertinib. Finally, we found that plasma PrPC levels are increased in EGFR-mutated NSCLC patients from 2 independent cohorts and that their longitudinal evolution mirrors that of disease. Altogether, these findings define PrPC as a candidate driver of EMT-dependent resistance to EGFR-TKI in NSCLC. They further suggest that monitoring plasma PrPC levels may represent a valuable non-invasive strategy for patient follow-up and warrant considering PrPC-targeted therapies for EGFR-mutated NSCLC patients with TKI failure.

Differential expression of cellular prion protein (PrPC) in mouse hepatitis virus induced neuroinflammation.

The cellular prion protein (PrPC) is an extracellular cell membrane protein. Due to its diversified roles, a definite role of PrPC has been difficult to establish. During viral infection, PrPC has been reported to play a pleiotropic role. Here, we have attempted to envision the function of PrPC in the neurotropic m-CoV-MHV-RSA59-induced model of neuroinflammation in C57BL/6 mice. A significant upregulation of PrPC at protein and mRNA levels was evident in infected mouse brains during the acute phase of neuroinflammation. Furthermore, investigation of the effect of MHV-RSA59 infection on PrPC expression in specific neuronal, microglial, and astrocytoma cell lines, revealed a differential expression of prion protein during neuroinflammation. Additionally, siRNA-mediated downregulation of prnp transcripts reduced the expression of viral antigen and viral infectivity in these cell lines. Cumulatively, our results suggest that PrPC expression significantly increases during acute MHV-RSA59 infection and that PrPC also assists in viral infectivity and viral replication.

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.

New Light on Prions: Putative Role of PrPc in Pathophysiology of Mood Disorders.

Mood disorders are highly prevalent and heterogenous mental illnesses with devastating rates of mortality and treatment resistance. The molecular basis of those conditions involves complex interplay between genetic and environmental factors. Currently, there are no objective procedures for diagnosis, prognosis and personalization of patients' treatment. There is an urgent need to search for novel molecular targets for biomarkers in mood disorders. Cellular prion protein (PrPc) is infamous for its potential to convert its insoluble form, leading to neurodegeneration in Creutzfeldt-Jacob disease. Meanwhile, in its physiological state, PrPc presents neuroprotective features and regulates neurotransmission and synaptic plasticity. The aim of this study is to integrate the available knowledge about molecular mechanisms underlying the impact of PrPc on the pathophysiology of mood disorders. Our review indicates an important role of this protein in regulation of cognitive functions, emotions, sleep and biological rhythms, and its deficiency results in depressive-like behavior and cognitive impairment. PrPc plays a neuroprotective role against excitotoxicity, oxidative stress and inflammation, the main pathophysiological events in the course of mood disorders. Research indicates that PrPc may be a promising biomarker of cognitive decline. There is an urgent need of human studies to elucidate its potential utility in clinical practice.

Application of N-Terminal Labeling Methods Provide Novel Insights into Endoproteolysis of the Prion Protein in Vivo.

Alternative α- and ß-cleavage events in the cellular prion protein (PrPC) central region generate fragments with distinct biochemical features that affect prion disease pathogenesis, but the assignment of precise cleavage positions has proven challenging. Exploiting mouse transgenic models expressing wild-type (WT) PrPC and an octarepeat region mutant allele (S3) with increased ß-fragmentation, cleavage sites were defined using LC-MS/MS in conjunction with N-terminal enzymatic labeling and chemical in-gel acetylation. Our studies profile the net proteolytic repertoire of the adult brain, as deduced from defining hundreds of proteolytic events in other proteins, and position individual cleavage events in PrPC α- and ß-target areas imputed from earlier, lower resolution methods; these latter analyses established site heterogeneity, with six cleavage sites positioned in the ß-cleavage region of WT PrPC and nine positions for S3 PrPC. Regarding α-cleavage, aside from reported N-termini at His110 and Val111, we identified a total of five shorter fragments in the brain of both mice lines. We infer that aminopeptidase activity in the brain could contribute to the ragged N-termini observed around PrPC's α- and ß-cleavage sites, with this work providing a point of departure for further in vivo studies of brain proteases.

Channel Activities of the Full-Length Prion and Truncated Proteins.

Prion diseases are fatal neurodegenerative disorders characterized by the conversion of the cellular prion protein (PrPC) into a misfolded prion form, which is believed to disrupt the cellular membranes. However, the exact mechanisms underlying prion toxicity, including the formation of membrane pores, are not fully understood. The prion protein consists of two domains: a globular domain (GD) and a flexible N-terminus (FT) domain. Although a proximal polybasic amino acid (FT(23-31) sequence of FT is a prerequisite for cellular membrane permeabilization, other functional domain regions may modulate its effects. Through single-channel electrical recordings and cryo-electron microscopy (cryo-EM), we discovered that the FT(23-50) fragment forms pore-shaped oligomers and plays a dominant role in membrane permeabilization within the full-length mouse prion protein (mPrP(23-230)). In contrast, the FT(51-110) domain or the C-terminal domain downregulate the channel activity of FT(23-50) and mPrP(23-230). The addition of prion mimetic antibody, POM1 significantly amplifies mPrP(23-230) membrane permeabilization, whereas POM1_Y104A, a mutant that binds to PrP but cannot elicit toxicity, has a negligible effect on membrane permeabilization. Additionally, the anti-N-terminal antibody POM2 or Cu2+ binds to the FT domain, subsequently enhancing the FT(23-110) channel activity. Importantly, our setup provides a novel approach without an external fused protein to examine the channel activity of truncated PrP in the lipid membranes. We therefore propose that the primary N-terminal residues are essential for membrane permeabilization, while other functional segments of PrP play a vital role in modulating the pathological effects of PrP-mediated neurotoxicity.

Excess PrPC inhibits muscle cell differentiation via miRNA-enhanced liquid-liquid phase separation implicated in myopathy.

The cellular prion protein (PrPC) is required for skeletal muscle function. Here, we report that a higher level of PrPC accumulates in the cytoplasm of the skeletal muscle of six myopathy patients compared to controls. PrPC inhibits skeletal muscle cell autophagy, and blocks myoblast differentiation. PrPC selectively binds to a subset of miRNAs during myoblast differentiation, and the colocalization of PrPC and miR-214-3p was observed in the skeletal muscle of six myopathy patients with excessive PrPC. We demonstrate that PrPC is overexpressed in skeletal muscle cells under pathological conditions, inhibits muscle cell differentiation by physically interacting with a subset of miRNAs, and selectively recruits these miRNAs into its phase-separated condensate in living myoblasts, which in turn enhances liquid-liquid phase separation of PrPC, promotes pathological aggregation of PrP, and results in the inhibition of autophagy-related protein 5-dependent autophagy and muscle bundle formation in myopathy patients characterized by incomplete muscle regeneration.

Two mouse models of Alzheimer's disease accumulate amyloid at different rates and have distinct Aß oligomer profiles unaltered by ablation of cellular prion protein.

Oligomers formed from monomers of the amyloid ß-protein (Aß) are thought to be central to the pathogenesis of Alzheimer's disease (AD). Unsurprisingly for a complex disease, current mouse models of AD fail to fully mimic the clinical disease in humans. Moreover, results obtained in a given mouse model are not always reproduced in a different model. Cellular prion protein (PrPC) is now an established receptor for Aß oligomers. However, studies of the Aß-PrPC interaction in different mouse models have yielded contradictory results. Here we performed a longitudinal study assessing a range of biochemical and histological features in the commonly used J20 and APP-PS1 mouse models. Our analysis demonstrated that PrPC ablation had no effect on amyloid accumulation or oligomer production. However, we found that APP-PS1 mice had higher levels of oligomers, that these could bind to recombinant PrPC, and were recognised by the OC antibody which distinguishes parallel, in register fibrils. On the other hand, J20 mice had a lower level of Aß oligomers, which did not interact with PrPC when tested in vitro and were OC-negative. These results suggest the two mouse models produce diverse Aß assemblies that could interact with different targets, highlighting the necessity to characterise the conformation of the Aß oligomers concomitantly with the toxic cascade elicited by them. Our results provide an explanation for the apparent contradictory results found in APP-PS1 mice and the J20 mouse line in regards to Aß toxicity mediated by PrPC.

Emerging roles of the cellular prion protein (PrPC) and 37/67 kDa laminin receptor (RPSA) interaction in cancer biology.

The cellular prion protein (PrPC) is well-known for its involvement, under its pathogenic protease-resistant form (PrPSc), in a group of neurodegenerative diseases, known as prion diseases. PrPC is expressed in nervous system, as well as in other peripheral organs, and has been found overexpressed in several types of solid tumors. Notwithstanding, studies in recent years have disclosed an emerging role for PrPC in various cancer associated processes. PrPC has high binding affinity for 37/67 kDa laminin receptor (RPSA), a molecule that acts as a key player in tumorigenesis, affecting cell growth, adhesion, migration, invasion and cell death processes. Recently, we have characterized at cellular level, small molecules able to antagonize the direct PrPC binding to RPSA and their intracellular trafficking. These findings are very crucial considering that the main function of RPSA is to modulate key events in the metastasis cascade. Elucidation of the role played by PrPC/RPSA interaction in regulating tumor development, progression and response to treatment, represents a very promising challenge to gain pathogenetic information and discover novel specific biomarkers and/or therapeutic targets to be exploited in clinical settings. This review attempts to convey a detailed description of the complexity surrounding these multifaceted proteins from the perspective of cancer hallmarks, but with a specific focus on the role of their interaction in the control of proliferation, migration and invasion, genome instability and mutation, as well as resistance to cell death controlled by autophagic pathway.

Oral vaccination as a potential strategy to manage chronic wasting disease in wild cervid populations.

Prion diseases are a novel class of infectious disease based in the misfolding of the cellular prion protein (PrPC) into a pathological, self-propagating isoform (PrPSc). These fatal, untreatable neurodegenerative disorders affect a variety of species causing scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in cervids, and Creutzfeldt-Jacob disease (CJD) in humans. Of the animal prion diseases, CWD is currently regarded as the most significant threat due its ongoing geographical spread, environmental persistence, uptake into plants, unpredictable evolution, and emerging evidence of zoonotic potential. The extensive efforts to manage CWD have been largely ineffective, highlighting the need for new disease management tools, including vaccines. Development of an effective CWD vaccine is challenged by the unique biology of these diseases, including the necessity, and associated dangers, of overcoming immune tolerance, as well the logistical challenges of vaccinating wild animals. Despite these obstacles, there has been encouraging progress towards the identification of safe, protective antigens as well as effective strategies of formulation and delivery that would enable oral delivery to wild cervids. In this review we highlight recent strategies for antigen selection and optimization, as well as considerations of various platforms for oral delivery, that will enable researchers to accelerate the rate at which candidate CWD vaccines are developed and evaluated.

Peptide aptamer targeting Aß-PrP-Fyn axis reduces Alzheimer's disease pathologies in 5XFAD transgenic mouse model.

Currently, no effective therapeutics exist for the treatment of incurable neurodegenerative diseases such as Alzheimer's disease (AD). The cellular prion protein (PrPC) acts as a high-affinity receptor for amyloid beta oligomers (AßO), a main neurotoxic species mediating AD pathology. The interaction of AßO with PrPC subsequently activates Fyn tyrosine kinase and neuroinflammation. Herein, we used our previously developed peptide aptamer 8 (PA8) binding to PrPC as a therapeutic to target the AßO-PrP-Fyn axis and prevent its associated pathologies. Our in vitro results indicated that PA8 prevents the binding of AßO with PrPC and reduces AßO-induced neurotoxicity in mouse neuroblastoma N2a cells and primary hippocampal neurons. Next, we performed in vivo experiments using the transgenic 5XFAD mouse model of AD. The 5XFAD mice were treated with PA8 and its scaffold protein thioredoxin A (Trx) at a 14.4 µg/day dosage for 12 weeks by intraventricular infusion through Alzet® osmotic pumps. We observed that treatment with PA8 improves learning and memory functions of 5XFAD mice as compared to Trx-treated 5XFAD mice. We found that PA8 treatment significantly reduces AßO levels and Aß plaques in the brain tissue of 5XFAD mice. Interestingly, PA8 significantly reduces AßO-PrP interaction and its downstream signaling such as phosphorylation of Fyn kinase, reactive gliosis as well as apoptotic neurodegeneration in the 5XFAD mice compared to Trx-treated 5XFAD mice. Collectively, our results demonstrate that treatment with PA8 targeting the AßO-PrP-Fyn axis is a promising and novel approach to prevent and treat AD.

Melatonin-Assisted Cisplatin Suppresses Urinary Bladder Cancer Cell Proliferation and Growth through Inhibiting PrPC-Regulated Cell Stress and Cell Proliferation Signaling.

This study investigated whether melatonin (Mel) would promote cisplatin to suppress the proliferation and growth of bladder cancer (BC) cells by inhibiting cellular prion protein (PrPC)-mediated cell stress and cell proliferation signaling. An immunohistochemical staining of tissue arrays from BC patients demonstrated that the PrPC expression was significantly upregulated from stage I to III BC (p < 0.0001). The BC cellline of T24 was categorized into G1 (T24), G2 (T24 + Mel/100 µM), G3 (T24+cisplatin/6 µM), G4 (PrPC overexpression in T24 (i.e., PrPC-OE-T24)), G5 (PrPC-OE-T24+Mel), and G6 (PrPC-OE-T24+cisplatin). When compared with a human uroepithelial cell line (SV-HUC-1), the cellular viability/wound healing ability/migration rate were significantly increased in T24 cells (G1) and further significantly increased in PrPC-OE-T24 cells (G4); and they were suppressed in Mel (G2/G5) or cisplatin (G3/G6) treatment (all p < 0.0001). Additionally, the protein expressions of cell proliferation (PI3K/p-Akt/p-m-TOR/MMP-9/PrPC), cell cycle/mitochondrial functional integrity (cyclin-D1/clyclin-E1/ckd2/ckd4/mitochondrial-cytochrome-C/PINK1), and cell stress (RAS/c-RAF/p-MEK1/2, p-ERK1/2) markers showed a similar pattern of cell viability among the groups (all p < 0.001). After the BC cell line of UMUC3 was implanted into nude mouse backs, by day 28 mthe BC weight/volume and the cellular levels of PrPC/MMP-2/MMP-9 were significantly, gradually reduced from groups one to four (all p < 0.0001). The protein expressions of cell proliferation (PI3K/p-Akt/p-m-TOR/MMP-9/PrPC), cell cycle/mitophagy (cyclin-D1/clyclin-E1/ckd2/ckd4/PINK1), and cell stress (RAS/c-RAF/p-MEK1,2/p-ERK1,2) signaling were significantly, progressively reduced from groups one to four, whereas the protein expressions of apoptotic (Mit-Bax/cleaved-caspase-3/cleaved-PARP) and oxidative stress/mitochondrial damaged (NOX-1/NOX-2/cytosolic-cytochrome-C/p-DRP1) markers expressed an opposite pattern of cell proliferation signaling among the groups (all p < 0.0001). Mel-cisplatin suppressed BC cell growth/proliferation via inhibiting the PrPC in upregulating the cell proliferation/cell stress/cell cycle signaling.

The Role of Cellular Prion Protein in Glioma Tumorigenesis Could Be through the Autophagic Mechanisms: A Narrative Review.

The carcinogenesis of glial tumors appears complex because of the many genetic and epigenetic phenomena involved. Among these, cellular prion protein (PrPC) is considered a key factor in cell-death resistance and important aspect implicated in tumorigenesis. Autophagy also plays an important role in cell death in various pathological conditions. These two cellular phenomena are related and share the same activation by specific alterations in the cellular microenvironment. Furthermore, there is an interdependence between autophagy and prion activity in glioma tumorigenesis. Glioma is one of the most aggressive known cancers, and the fact that such poorly studied processes as autophagy and PrPC activity are so strongly involved in its carcinogenesis suggests that by better understanding their interaction, more can be understood about its origin and treatment. Few studies in the literature relate these two cellular phenomena, much less try to explain their combined activity and role in glioma carcinogenesis. In this study, we explored the recent findings on the molecular mechanism and regulation pathways of autophagy, examining the role of PrPC in autophagy processes and how they may play a central role in glioma tumorigenesis. Among the many molecular interactions that PrP physiologically performs, it appears that processes shared with autophagy activity are those most implicated in glial tumor carcinogeneses such as activity on MAP kinases, PI3K, and mTOR. This work can be supportive and valuable as a basis for further future studies on this topic.

Bifunctional carbazole derivatives for simultaneous therapy and fluorescence imaging in prion disease murine cell models.

Prion diseases are characterized by the self-assembly of pathogenic misfolded scrapie isoforms (PrPSc) of the cellular prion protein (PrPC). In an effort to achieve a theranostic profile, symmetrical bifunctional carbazole derivatives were designed as fluorescent rigid analogues of GN8, a pharmacological chaperone that stabilizes the native PrPC conformation and prevents its pathogenic conversion. A focused library was synthesized via a four-step route, and a representative member was confirmed to have native fluorescence, including a band in the near-infrared region. After a cytotoxicity study, compounds were tested on the RML-infected ScGT1 neuronal cell line, by monitoring the levels of protease-resistant PrPSc. Small dialkylamino groups at the ends of the molecule were found to be optimal in terms of therapeutic index, and the bis-(dimethylaminoacetamido)carbazole derivative 2b was selected for further characterization. It showed activity in two cell lines infected with the mouse-adapted RML strain (ScGT1 and ScN2a). Unlike GN8, 2b did not affect PrPC levels, which represents a potential advantage in terms of toxicity. Amyloid Seeding Assay (ASA) experiments showed the capacity of 2b to delay the aggregation of recombinant mouse PrP. Its ability to interfere with the amplification of the scrapie RML strain by Protein Misfolding Cyclic Amplification (PMCA) was shown to be higher than that of GN8, although 2b did not inhibit the amplification of human vCJD prion. Fluorescent staining of PrPSc aggregates by 2b was confirmed in living cells. 2b emerges as an initial hit compound for further medicinal chemistry optimization towards strain-independent anti-prion compounds.

Beta-endoproteolysis of the cellular prion protein by dipeptidyl peptidase-4 and fibroblast activation protein.

The cellular prion protein (PrPC) converts to alternatively folded pathogenic conformations (PrPSc) in prion infections and binds neurotoxic oligomers formed by amyloid-ß α-synuclein, and tau. ß-Endoproteolysis, which splits PrPC into N- and C-terminal fragments (N2 and C2, respectively), is of interest because a protease-resistant, C2-sized fragment (C2Sc) accumulates in the brain during prion infections, seemingly comprising the majority of PrPSc at disease endpoint in mice. However, candidates for the underlying proteolytic mechanism(s) remain unconfirmed in vivo. Here, a cell-based screen of protease inhibitors unexpectedly linked type II membrane proteins of the S9B serine peptidase subfamily to PrPC ß-cleavage. Overexpression experiments in cells and assays with recombinant proteins confirmed that fibroblast activation protein (FAP) and its paralog, dipeptidyl peptidase-4 (DPP4), cleave directly at multiple sites within PrPC's N-terminal domain. For wild-type mouse and human PrPC substrates expressed in cells, the rank orders of activity were human FAP ~ mouse FAP > mouse DPP4 > human DPP4 and human FAP > mouse FAP > mouse DPP4 >> human DPP4, respectively. C2 levels relative to total PrPC were reduced in several tissues from FAP-null mice, and, while knockout of DPP4 lacked an analogous effect, the combined DPP4/FAP inhibitor linagliptin, but not the FAP-specific inhibitor SP-13786, reduced C2Sc and total PrPSc levels in two murine cell-based models of prion infections. Thus, the net activity of the S9B peptidases FAP and DPP4 and their cognate inhibitors/modulators affect the physiology and pathogenic potential of PrPC.

Cellular prion protein offers neuroprotection in astrocytes submitted to amyloid ß oligomer toxicity.

The cellular prion protein (PrPC), in its native conformation, performs numerous cellular and cognitive functions in brain tissue. However, despite the cellular prion research in recent years, there are still questions about its participation in oxidative and neurodegenerative processes. This study aims to elucidate the involvement of PrPC in the neuroprotection cascade in the presence of oxidative stressors. For that, astrocytes from wild-type mice and knockout to PrPC were subjected to the induction of oxidative stress with hydrogen peroxide (H2O2) and with the toxic oligomer of the amyloid ß protein (AßO). We observed that the presence of PrPC showed resistance in the cell viability of astrocytes. It was also possible to monitor changes in basic levels of metals and associate them with an induced damage condition, indicating the precise role of PrPC in metal homeostasis, where the absence of PrPC leads to metallic unbalance, culminating in cellular vulnerability to oxidative stress. Increased caspase 3, p-Tau, p53, and Bcl2 may establish a relationship between a PrPC and an induced damage condition. Complementarily, it has been shown that PrPC prevents the internalization of AßO and promotes its degradation under oxidative stress induction, thus preventing protein aggregation in astrocytes. It was also observed that the presence of PrPC can be related to translocating SOD1 to cell nuclei under oxidative stress, probably controlling DNA damage. The results of this study suggest that PrPC acts against oxidative stress activating the cellular response and defense by displaying neuroprotection to neurons and ensuring the functionality of astrocytes.

An optimized Western blot assay provides a comprehensive assessment of the physiological endoproteolytic processing of the prion protein.

The prion protein (PrPC) is subjected to several conserved endoproteolytic events producing bioactive fragments that are of increasing interest for their physiological functions and their implication in the pathogenesis of prion diseases and other neurodegenerative diseases. However, systematic and comprehensive investigations on the full spectrum of PrPC proteoforms have been hampered by the lack of methods able to identify all PrPC-derived proteoforms. Building on previous knowledge of PrPC endoproteolytic processing, we thus developed an optimized Western blot assay able to obtain the maximum information about PrPC constitutive processing and the relative abundance of PrPC proteoforms in a complex biological sample. This approach led to the concurrent identification of the whole spectrum of known endoproteolytic-derived PrPC proteoforms in brain homogenates, including C-terminal, N-terminal and, most importantly, shed PrPC-derived fragments. Endoproteolytic processing of PrPC was remarkably similar in the brain of widely used wild type and transgenic rodent models, with α-cleavage-derived C1 representing the most abundant proteoform and ADAM10-mediated shedding being an unexpectedly prominent proteolytic event. Interestingly, the relative amount of shed PrPC was higher in WT mice than in most other models. Our results indicate that constitutive endoproteolytic processing of PrPC is not affected by PrPC overexpression or host factors other than PrPC but can be impacted by PrPC primary structure. Finally, this method represents a crucial step in gaining insight into pathophysiological roles, biomarker suitability, and therapeutic potential of shed PrPC and for a comprehensive appraisal of PrPC proteoforms in therapies, drug screening, or in the progression of neurodegenerative diseases.

Development of Alkaline Phosphatase-Fused Mouse Prion Protein and Its Application in Toxic Aß Oligomer Detection.

Amyloid ß (Aß) oligomers play a key role in the progression of Alzheimer's disease (AD). Multiple forms of Aß assemblies have been identified by in vitro and in vivo analyses; however, it is uncertain which oligomer is highly neurotoxic. Thus, understanding the pathogenesis of AD by detecting toxic Aß oligomers is crucial. In this study, we report a fusion protein of cellular prion protein (PrPc) and alkaline phosphatase (ALP) from Escherichia coli as a sensing element for toxic Aß oligomers. Since the N-terminus domain of PrPc (residue 23-111) derived from mice is known to bind to toxic Aß oligomers in vitro, we genetically fused PrPc23-111 to ALP. The developed fusion protein, PrP-ALP, retained both the binding ability of PrPc and enzymatic activity of ALP. We showed that PrP-ALP strongly bound to high molecular weight (HMW) oligomers but showed little or no affinity toward monomers. The observation that PrP-ALP neutralized the toxic effect of Aß oligomers indicated an interaction between PrP-ALP and toxic HMW oligomers. Based on ALP activity, we succeeded in detecting Aß oligomers. PrP-ALP may serve as a powerful tool for detecting toxic Aß oligomers that may be related to AD progression.