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.

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.

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.

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.

The multiple functions of PrPC in physiological, cancer, and neurodegenerative contexts.

Cellular prion protein (PrPC) is a highly conserved glycoprotein, present both anchored in the cell membrane and soluble in the extracellular medium. It has a diversity of ligands and is variably expressed in numerous tissues and cell subtypes, most notably in the central nervous system (CNS). Its importance has been brought to light over the years both under physiological conditions, such as embryogenesis and immune system homeostasis, and in pathologies, such as cancer and neurodegenerative diseases. During development, PrPC plays an important role in CNS, participating in axonal growth and guidance and differentiation of glial cells, but also in other organs such as the heart, lung, and digestive system. In diseases, PrPC has been related to several types of tumors, modulating cancer stem cells, enhancing malignant properties, and inducing drug resistance. Also, in non-neoplastic diseases, such as Alzheimer's and Parkinson's diseases, PrPC seems to alter the dynamics of neurotoxic aggregate formation and, consequently, the progression of the disease. In this review, we explore in detail the multiple functions of this protein, which proved to be relevant for understanding the dynamics of organism homeostasis, as well as a promising target in the treatment of both neoplastic and degenerative diseases.

A conformational switch controlling the toxicity of the prion protein.

Prion infections cause conformational changes of the cellular prion protein (PrPC) and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond ('H-latch'), altering the flexibility of the α2-α3 and ß2-α2 loops of PrPC. Expression of a PrP2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrPR207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrPC, but not PrP2Cys. These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is an important reporter of prion neurotoxicity.

A Soluble PrPC Derivative and Membrane-Anchored PrPC in Extracellular Vesicles Attenuate Innate Immunity by Engaging the NMDA-R/LRP1 Receptor Complex.

Nonpathogenic cellular prion protein (PrPC) demonstrates anti-inflammatory activity; however, the responsible mechanisms are incompletely defined. PrPC exists as a GPI-anchored membrane protein in diverse cells; however, PrPC may be released from cells by ADAM proteases or when packaged into extracellular vesicles (EVs). In this study, we show that a soluble derivative of PrPC (S-PrP) counteracts inflammatory responses triggered by pattern recognition receptors in macrophages, including TLR2, TLR4, TLR7, TLR9, NOD1, and NOD2. S-PrP also significantly attenuates the toxicity of LPS in mice. The response of macrophages to S-PrP is mediated by a receptor assembly that includes the N-methyl-d-aspartate receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1). PrPC was identified in EVs isolated from human plasma. These EVs replicated the activity of S-PrP, inhibiting cytokine expression and IκBα phosphorylation in LPS-treated macrophages. The effects of plasma EVs on LPS-treated macrophages were blocked by PrPC-specific Ab, by antagonists of LRP1 and the NMDA-R, by deleting Lrp1 in macrophages, and by inhibiting Src family kinases. Phosphatidylinositol-specific phospholipase C dissociated the LPS-regulatory activity from EVs, rendering the EVs inactive as LPS inhibitors. The LPS-regulatory activity that was lost from phosphatidylinositol-specific phospholipase C-treated EVs was recovered in solution. Collectively, these results demonstrate that GPI-anchored PrPC is the essential EV component required for the observed immune regulatory activity of human plasma EVs. S-PrP and EV-associated PrPC regulate innate immunity by engaging the NMDA-R/LRP1 receptor system in macrophages. The scope of pattern recognition receptors antagonized by S-PrP suggests that released forms of PrPC may have broad anti-inflammatory activity.

Virus Infection, Genetic Mutations, and Prion Infection in Prion Protein Conversion.

Conformational conversion of the cellular isoform of prion protein, PrPC, into the abnormally folded, amyloidogenic isoform, PrPSc, is an underlying pathogenic mechanism in prion diseases. The diseases manifest as sporadic, hereditary, and acquired disorders. Etiological mechanisms driving the conversion of PrPC into PrPSc are unknown in sporadic prion diseases, while prion infection and specific mutations in the PrP gene are known to cause the conversion of PrPC into PrPSc in acquired and hereditary prion diseases, respectively. We recently reported that a neurotropic strain of influenza A virus (IAV) induced the conversion of PrPC into PrPSc as well as formation of infectious prions in mouse neuroblastoma cells after infection, suggesting the causative role of the neuronal infection of IAV in sporadic prion diseases. Here, we discuss the conversion mechanism of PrPC into PrPSc in different types of prion diseases, by presenting our findings of the IAV infection-induced conversion of PrPC into PrPSc and by reviewing the so far reported transgenic animal models of hereditary prion diseases and the reverse genetic studies, which have revealed the structure-function relationship for PrPC to convert into PrPSc after prion infection.

The protease-sensitive N-terminal polybasic region of prion protein modulates its conversion to the pathogenic prion conformer.

Conversion of normal prion protein (PrPC) to the pathogenic PrPSc conformer is central to prion diseases such as Creutzfeldt-Jakob disease and scrapie; however, the detailed mechanism of this conversion remains obscure. To investigate how the N-terminal polybasic region of PrP (NPR) influences the PrPC-to-PrPSc conversion, we analyzed two PrP mutants: ΔN6 (deletion of all six amino acids in NPR) and Met4-1 (replacement of four positively charged amino acids in NPR with methionine). We found that ΔN6 and Met4-1 differentially impacted the binding of recombinant PrP (recPrP) to the negatively charged phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, a nonprotein cofactor that facilitates PrP conversion. Both mutant recPrPs were able to form recombinant prion (recPrPSc) in vitro, but the convertibility was greatly reduced, with ΔN6 displaying the lowest convertibility. Prion infection assays in mammalian RK13 cells expressing WT or NPR-mutant PrPs confirmed these differences in convertibility, indicating that the NPR affects the conversion of both bacterially expressed recPrP and post-translationally modified PrP in eukaryotic cells. We also found that both WT and mutant recPrPSc conformers caused prion disease in WT mice with a 100% attack rate, but the incubation times and neuropathological changes caused by two recPrPSc mutants were significantly different from each other and from that of WT recPrPSc. Together, our results support that the NPR greatly influences PrPC-to-PrPSc conversion, but it is not essential for the generation of PrPSc. Moreover, the significant differences between ΔN6 and Met4-1 suggest that not only charge but also the identity of amino acids in NPR is important to PrP conversion.

The Cellular Prion Protein Increases the Uptake and Toxicity of TDP-43 Fibrils.

Cytoplasmic aggregation of the primarily nuclear TAR DNA-binding protein 43 (TDP-43) affects neurons in most amyotrophic lateral sclerosis (ALS) and approximately half of frontotemporal lobar degeneration (FTLD) cases. The cellular prion protein, PrPC, has been recognized as a common receptor and downstream effector of circulating neurotoxic species of several proteins involved in neurodegeneration. Here, capitalizing on our recently adapted TDP-43 real time quaking induced reaction, we set reproducible protocols to obtain standardized preparations of recombinant TDP-43 fibrils. We then exploited two different cellular systems (human SH-SY5Y and mouse N2a neuroblastoma cells) engineered to express low or high PrPC levels to investigate the link between PrPC expression on the cell surface and the internalization of TDP-43 fibrils. Fibril uptake was increased in cells overexpressing either human or mouse prion protein. Increased internalization was associated with detrimental consequences in all PrP-overexpressing cell lines but was milder in cells expressing the human form of the prion protein. As described for other amyloids, treatment with TDP-43 fibrils induced a reduction in the accumulation of the misfolded form of PrPC, PrPSc, in cells chronically infected with prions. Our results expand the list of misfolded proteins whose uptake and detrimental effects are mediated by PrPC, which encompass almost all pathological amyloids involved in neurodegeneration.

Potential for transmission of sporadic Creutzfeldt-Jakob disease through peripheral routes.

Five sporadic Creutzfeldt-Jakob disease (CJD) strains have been identified to date, based on differences in clinicopathological features of the patients, the biochemical properties of abnormal prion proteins, and transmission properties. Recent advances in our knowledge about iatrogenic transmission of sporadic CJD have raised the possibility that the infectivity of sporadic CJD strains through peripheral routes is different from that of intracranial infection. To test this possibility, here we assessed systematically the infectivity of sporadic CJD strains through the peripheral route for the first time using a mouse model expressing human prion protein. Although the infectivity of the V2 and M1 sporadic CJD strains is almost the same in intracerebral transmission studies, the V2 strain infected more efficiently than the M1 strain through the peripheral route. The other sporadic CJD strains examined lacked infectivity. Of note, both the V2 and M1 strains showed preference for mice with the valine homozygosity at the PRNP polymorphic codon. These results indicate that the V2 strain is the most infectious sporadic CJD strain for infection through peripheral routes. In addition, these findings raise the possibility that individuals with the valine homozygosity at the PRNP polymorphic codon might have higher risks of infection through peripheral routes compared with the methionine homozygotes. Thus, preventive measures against the transmission of the V2 sporadic CJD strain will be important for the eradication of iatrogenic CJD transmission through peripheral routes.

Altered cellular localisation and expression, together with unconventional protein trafficking, of prion protein, PrPC, in type 1 diabetes.

AIMS/HYPOTHESIS: Normal cellular prion protein (PrPC) is a conserved mammalian glycoprotein found on the outer plasma membrane leaflet through a glycophosphatidylinositol anchor. Although PrPC is expressed by a wide range of tissues throughout the body, the complete repertoire of its functions has not been fully determined. The misfolded pathogenic isoform PrPSc (the scrapie form of PrP) is a causative agent of neurodegenerative prion diseases. The aim of this study is to evaluate PrPC localisation, expression and trafficking in pancreases from organ donors with and without type 1 diabetes and to infer PrPC function through studies on interacting protein partners. METHODS: In order to evaluate localisation and trafficking of PrPC in the human pancreas, 12 non-diabetic, 12 type 1 diabetic and 12 autoantibody-positive organ donor tissue samples were analysed using immunofluorescence analysis. Furthermore, total RNA was isolated from 29 non-diabetic, 29 type 1 diabetic and 24 autoantibody-positive donors to estimate PrPC expression in the human pancreas. Additionally, we performed PrPC-specific immunoblot analysis on total pancreatic protein from non-diabetic and type 1 diabetic organ donors to test whether changes in PrPC mRNA levels leads to a concomitant increase in PrPC protein levels in human pancreases. RESULTS: In non-diabetic and type 1 diabetic pancreases (the latter displaying both insulin-positive [INS(+)] and -negative [INS(-)] islets), we found PrPC in islets co-registering with beta cells in all INS(+) islets and, strikingly, unexpected activation of PrPC in alpha cells within diabetic INS(-) islets. We found PrPC localised to the plasma membrane and endoplasmic reticulum (ER) but not the Golgi, defining two cellular pools and an unconventional protein trafficking mechanism bypassing the Golgi. We demonstrate PrPC co-registration with established protein partners, neural cell adhesion molecule 1 (NCAM1) and stress-inducible phosphoprotein 1 (STI1; encoded by STIP1) on the plasma membrane and ER, respectively, linking PrPC function with cyto-protection, signalling, differentiation and morphogenesis. We demonstrate that both PRNP (encoding PrPC) and STIP1 gene expression are dramatically altered in type 1 diabetic and autoantibody-positive pancreases. CONCLUSIONS/INTERPRETATION: As the first study to address PrPC expression in non-diabetic and type 1 diabetic human pancreas, we provide new insights for PrPC in the pathogenesis of type 1 diabetes. We evaluated the cell-type specific expression of PrPC in the human pancreas and discovered possible connections with potential interacting proteins that we speculate might address mechanisms relevant to the role of PrPC in the human pancreas.

Mutation-Dependent Refolding of Prion Protein Unveils Amyloidogenic-Related Structural Ramifications: Insights from Molecular Dynamics Simulations.

The main focus of prion structural biology studies is to understand the molecular basis of prion diseases caused by misfolding, and aggregation of the cellular prion protein PrPC remains elusive. Several genetic mutations are linked with human prion diseases and driven by the conformational conversion of PrPC to the toxic PrPSc. The main goal of this study is to gain a better insight into the molecular effect of disease-associated V210I mutation on this process by molecular dynamics simulations. This inherited mutation elicited copious structural changes in the ß1-α1-ß2 subdomain, including an unfolding of a helix α1 and the elongation of the ß-sheet. These unusual structural changes likely appeared to detach the ß1-α1-ß2 subdomain from the α2-α3 core, an early misfolding event necessary for the conformational conversion of PrPC to PrPSc. Ultimately, the unfolded α1 and its prior ß1-α1 loop further engaged with unrestrained conformational dynamics and were widely considered as amyloidogenic-inducing traits. Furthermore, the resulting folding intermediate possesses a highly unstable ß1-α1-ß2 subdomain, thereby enhancing the aggregation of misfolded PrPC through intermolecular interactions between frequently refolding regions. Briefly, these remarkable changes as seen in the mutant ß1-α1-ß2 subdomain are consistent with previous experimental results and thus provide a molecular basis of PrPC misfolding associated with the conformational conversion of PrPC to PrPSc.

From Kuru to Alzheimer: A personal outlook.

Seventy years ago, we learned from Chris Anfinsen that the stereochemical code necessary to fold a protein is embedded into its amino acid sequence. In water, protein morphogenesis is a spontaneous reversible process leading from an ensemble of disordered structures to the ordered functionally competent protein; conforming to Aristotle's definition of substance, the synolon of matter and form. The overall process of folding is generally consistent with a two state transition between the native and the denatured protein: not only the denatured state is an ensemble of several structures, but also the native protein populates distinct functionally relevant conformational (sub)states. This two-state view should be revised, given that any globular protein can populate a peculiar third state called amyloid, characterized by an overall architecture that at variance with the native state, is by-and-large independent of the primary structure. In a nut shell, we should accept that beside the folded and unfolded states, any protein can populate a third state called amyloid which gained center stage being the hallmark of incurable neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases as well as others. These fatal diseases are characterized by clear-cut clinical differences, yet display some commonalities such as the presence in the brain of amyloid deposits constituted by one misfolded protein specific for each disease. Some aspects of this complex problem are summarized here as an excursus from the prion's fibrils observed in the brain of aborigines who died of Kuru to the amyloid detectable in the cortex of Alzheimer's patients.