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The cellular prion protein (PrP(C)) is comprised of two domains – a globular C-terminal domain and an unstructured N-terminal domain. Recently, copper has been observed to drive tertiary contact in PrP(C), inducing a neuroprotective cis interaction that structurally links the protein’s two domains. The location of this interaction on the C-terminus overlaps with the sites of human pathogenic mutations and toxic antibody docking. Combined with recent evidence that the N-terminus is a toxic effector regulated by the C-terminus, there is an emerging consensus that this cis interaction serves a protective role, and that the disruption of this interaction by misfolded PrP oligomers may be a cause of toxicity in prion disease. We demonstrate here that two highly conserved histidines in the C-terminal domain of PrP(C) are essential for the protein’s cis interaction, which helps to protect against neurotoxicity carried out by its N-terminus. We show that simultaneous mutation of these histidines drastically weakens the cis interaction and enhances spontaneous cationic currents in cultured cells - the first C-terminal mutant to do so. Whereas previous studies suggested that Cu(2+) coordination was localized solely to the protein’s N-terminal domain, we find that both domains contribute equatorially coordinated histidine residue side chains, resulting in a novel bridging interaction. We also find that extra N-terminal histidines in pathological familial mutations involving octarepeat expansions inhibit this interaction by sequestering copper from the C-terminus. Our findings further establish a structural basis for PrP(C)’s C-terminal regulation of its otherwise toxic N-terminus.

Alzheimer’s disease (AD) is the main cause of dementia, and β‐amyloid (Aβ) is a central factor in the initiation and progression of the disease. Different forms of Aβ have been identified as monomers, oligomers, and amyloid fibrils. Many proteins have been implicated as putative receptors of respective forms of Aβ. Distinct forms of Aβ oligomers are considered to be neurotoxic species that trigger the pathophysiology of AD. It was reported that cellular prion protein (PrPC) is one of the most selective and high‐affinity binding partners of Aβ oligomers. The interaction of Aβ oligomers with PrPC is important to synaptic dysfunction and loss. The binding of Aβ oligomers to PrPC has mostly been studied with synthetic peptides, cell culture, and murine models of AD by biochemical and biological methods. However, the molecular mechanisms underlying the relationship between Aβ oligomers and PrPC remain unclear, especially in the human brain. We immunohistochemically investigated the relationship between Aβ oligomers and PrPC in human brain tissue with and without amyloid pathology. We histologically demonstrate that PrPC accumulates with aging in human brain tissue even prior to AD mainly within diffuse‐type amyloid plaques, which are composed of more soluble Aβ oligomers without stacked β‐sheet fibril structures. Our results suggest that PrPC accumulating plaques are associated with more soluble Aβ oligomers, and appear even prior to AD. The investigation of PrPC accumulating plaques may provide new insights into AD., PrPC accumulates within a subset of diffuse‐type amyloid plaques (arrowheads). We propose this new subtype of amyloid plaque as the PrP (+) plaque, composed of more soluble and oligomeric Aβ. Antibodies: 6E10 (left), 3F4 (right); Insets: Higher magnification of the plaques (*).

Decades of research efforts have conclusively provided overwhelming evidence that the cellular prion protein (PrPC) plays a central role in prion diseases, a set of fatal and incurable neurodegenerative disorders for which no therapy is yet available. In this Viewpoint, we provide an overview of the drug discovery strategies in the field, highlighting the current therapeutic hypotheses targeting, whether directly or indirectly, PrPC as well as the antiprion agents closest to clinical application.

Myelin is a dynamic membrane that is important for coordinating the fast propagation of action potentials along small or large caliber axons (0.1–10 μm) some of which extend the entire length of the spinal cord. Due to the heterogeneity of electrical and energy demands of the variable neuronal populations, the axo-myelinic and axo-glial interactions that regulate the biophysical properties of myelinated axons also vary in terms of molecular interactions at the membrane interfaces. An important topic of debate in neuroscience is how myelin is maintained and modified under neuronal control and how disruption of this control (due to disease or injury) can initiate and/or propagate neurodegeneration. One of the key molecular signaling cascades that have been investigated in the context of neural injury over the past two decades involves the myelin-associated inhibitory factors (MAIFs) that interact with Nogo receptor 1 (NgR1). Chief among the MAIF superfamily of molecules is a reticulon family protein, Nogo-A, that is established as a potent inhibitor of neurite sprouting and axon regeneration. However, an understated role for NgR1 is its ability to control axo-myelin interactions and Nogo-A specific ligand binding. These interactions may occur at axo-dendritic and axo-glial synapses regulating their functional and dynamic membrane domains. The current review provides a comprehensive analysis of how neuronal NgR1 can regulate myelin thickness and plasticity under normal and disease conditions. Specifically, we discuss how NgR1 plays an important role in regulating paranodal and juxtaparanodal domains through specific signal transduction cascades that are important for microdomain molecular architecture and action potential propagation. Potential therapeutics designed to target NgR1-dependent signaling during disease are being developed in animal models since interference with the involvement of the receptor may facilitate neurological recovery. Hence, the regulatory role played by NgR1 in the axo-myelinic interface is an important research field of clinical significance that requires comprehensive investigation.

Calcium (Ca2+) is an intracellular second messenger that ubiquitously masters remarkably diverse biological processes, including cell death. Growing evidence substantiates an involvement of the prion protein (PrPC) in regulating neuronal Ca2+ homeostasis, which could rationalize most of the wide range of functions ascribed to the protein. We have recently demonstrated that PrPC controls extracellular Ca2+ fluxes, and mitochondrial Ca2+ uptake, in neurons stimulated with glutamate (De Mario et al., J Cell Sci 2017; 130:2736-46), suggesting that PrPC protects neurons from threatening Ca2+ overloads and excitotoxicity. In light of these results and of recent reports in the literature, here we review the connection of PrPC with Ca2+ metabolism and also provide some speculative hints on the physiologic outcomes of this link. In addition, because PrPC is implicated in neurodegenerative diseases, including prion disorders and Alzheimer's disease, we will also discuss possible ways by which disruption of ...

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.

In prion diseases, the spread of infectious prions (PrPSc) is thought to occur within nerves and across synapses of the central nervous system (CNS). However, the mechanisms by which PrPSc moves within axons and across nerve synapses remain undetermined. Molecular motors, including kinesins and dyneins, transport many types of intracellular cargo. Kinesin-1C (KIF5C) has been shown to transport vesicles carrying the normal prion protein (PrPC) within axons, but whether KIF5C is involved in PrPSc axonal transport is unknown. The current study tested whether stereotactic inoculation in the striatum of KIF5C knock-out mice (Kif5c−/−) with 0.5 µL volumes of mouse-adapted scrapie strains 22 L or ME7 would result in an altered rate of prion spreading and/or disease timing. Groups of mice injected with each strain were euthanized at either pre-clinical time points or following the development of prion disease. Immunohistochemistry for PrP was performed on brain sections and PrPSc distribution and tempo of spread were compared between mouse strains. In these experiments, no differences in PrPSc spread, distribution or survival times were observed between C57BL/6 and Kif5c−/− mice.

A cellular prion protein (PrPC) is a ubiquitous cell surface glycoprotein, and its physiological functions have been receiving increased attention. Endogenous PrPC is present in various kidney tissues and undergoes glomerular filtration. In prion diseases, abnormal prion proteins are found to accumulate in renal tissues and filtered into urine. Urinary prion protein could serve as a diagnostic biomarker. PrPC plays a role in cellular signaling pathways, reno-protective effects, and kidney iron uptake. PrPC signaling affects mitochondrial function via the ERK pathway and is affected by the regulatory influence of microRNAs, small molecules, and signaling proteins. Targeting PrPC in acute and chronic kidney disease could help improve iron homeostasis, ameliorate damage from ischemia/reperfusion injury, and enhance the efficacy of mesenchymal stem/stromal cell or extracellular vesicle-based therapeutic strategies. PrPC may also be under the influence of BMP/Smad signaling and affect the progression of TGF-β-related renal fibrosis. PrPC conveys TNF-α resistance in some renal cancers, and therefore, the coadministration of anti-PrPC antibodies improves chemotherapy. PrPC can be used to design antibody–drug conjugates, aptamer–drug conjugates, and customized tissue inhibitors of metalloproteinases to suppress cancer. With preclinical studies demonstrating promising results, further research on PrPC in the kidney may lead to innovative PrPC-based therapeutic strategies for renal disease.

Prion diseases, or transmissible spongiform encephalopathies, have revealed the bewildering phenomenon of transmissibility in neurodegenerative diseases. Hence, the experimental transmissibility of prion-like neurodegenerative diseases via template directed misfolding has become the focus of intense research. Gerstmann-Sträussler-Scheinker disease (GSS) is an inherited prion disease associated with mutations in the prion protein gene. However, with the exception of a few GSS cases with P102L mutation characterized by co-accumulation of protease-resistant PrP core (PrPres) of ∼21 kDa, attempts to transmit to rodents GSS associated to atypical misfolded prion protein with ∼8 kDa PrPres have been unsuccessful. As a result, these GSS subtypes have often been considered as non-transmissible proteinopathies rather than true prion diseases. In a recent study we inoculated bank voles with GSS cases associated with P102L, A117V and F198S mutations and found that they transmitted efficiently and produced distinct pathological phenotypes, irrespective of the presence of 21 kDa PrPres in the inoculum. This study demonstrates that GSS is a genuine prion disease characterized by both transmissibility and strain variation. We discuss the implications of these findings for the understanding of the heterogeneous clinic-pathological phenotypes of GSS and of the molecular underpinnings of prion infectivity.

An unusual molecular structure of the prion protein, PrPsc is found only in mammals with transmissible prion diseases. Prion protein stands for either the infectious pathogen itself or a main component of it. Recent studies suggest that autophagy is one of the major functions that keep cells alive and has a protective effect against the neurodegeneration. In this study, we investigated that the effect of human prion protein on autophagy-lysosomal system of primary neuronal cells. The treatment of human prion protein induced primary neuron cell death and decreased both LC3-II and p62 protein amount indicating autophagy flux activation. Electron microscope pictures confirmed the autophagic flux activation in neuron cells treated with prion protein. Inhibition of autophagy flux using pharmacological and genetic tools prevented neuron cell death induced by human prion protein. Autophagy flux induced by prion protein is more activated in prpc expressing cells than in prpc silencing cells. These data demonstrated that prion protein-induced autophagy flux is involved in neuron cell death in prion disease and suggest that autophagy flux might play a critical role in neurodegenerative diseases including prion disease.

// Jae-Kyo Jeong 1,2 and Sang-Youel Park 1,2 1 Biosafety Research Institute, College of Veterinary Medicine, Chonbuk National University, Jeonju, Korea 2 Department of Bioactive Material Sciences and Research Center of Bioactive Materials, Chonbuk National University, Jeonju, Korea Correspondence to: Sang-Youel Park, email: // Keywords : autophagy flux, alpha-7 nicotinic receptor, prpc, prion, Geotarget Received : April 03, 2015 Accepted : June 28, 2015 Published : July 22, 2015 Abstract Activation of the alpha7 nicotinic acetylcholine receptor (α7nAchR ) is regulated by prion protein (PrPC) expression and has a neuroprotective effect by modulating autophagic flux. In this study, we hypothesized that PrPC may regulate α7nAchR activation and that may prevent prion-related neurodegenerative diseases by regulating autophagic flux. PrP(106–126) treatment decreased α 7nAchR expression and activation of autophagic flux. In addition, the α7nAchR activator PNU-282987 enhanced autophagic flux and protected neuron cells against PrP(106–126)-induced apoptosis. However, activation of autophagy and the protective effects of PNU-282987 were inhibited in PrPC knockout hippocampal neuron cells. In addition, PrPC knockout hippocampal neuron cells showed decreased α7nAchR expression levels. Adenoviral overexpression of PrPC in PrPC knockout hippocampal neuron cells resulted in activation of autophagic flux and inhibition of prion peptide-mediated cell death via α7nAchR activation. This is the first report demonstrating that activation of α7nAchR -mediated autophagic flux is regulated by PrPC, and that activation of α7nAchR regulated by PrPC expression may play a pivotal role in protection of neuron cells against prion peptide-induced neuron cell death by autophagy. These results suggest that α7nAchR -mediated autophagic flux may be involved in the pathogenesis of prion-related diseases and may be a therapeutic target for prion-related neurodegenerative diseases.

// Jin-Young Park 1, * , Jae-Kyo Jeong 1, 2, * , Ju-Hee Lee 1, 2 , Ji-Hong Moon 1, 2 , Sung-Wook Kim 1 , You-Jin Lee 1, 2 , Sang-Youel Park 1, 2 1 Biosafety Research Institute, College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk, South Korea 2 Department of Bioactive Material Sciences and Research Center of Bioactive Materials, Chonbuk National University, Jeonju, Jeonbuk, South Korea * These authors have contributed equally to this work Correspondence to: Sang-Youel Park, e-mail: sypark@chonbuk.ac.kr Keywords: PrPc, Hypoxia, TRAIL, HIF-1α, Colon cancer Received: October 01, 2014 Accepted: January 01, 2015 Published: January 20, 2015 ABSTRACT Hypoxia decreases cytotoxic responses to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein. Cellular prion protein (PrPc) is regulated by HIF-1α in neurons. We hypothesized that PrPc is involved in hypoxia-mediated resistance to TRAIL-induced apoptosis. We found that hypoxia induced PrPc protein and inhibited TRAIL-induced apoptosis. Thus silencing of PrPc increased TRAIL-induced apoptosis under hypoxia. Overexpression of PrPc protein using an adenoviral vector inhibited TRAIL-induced apoptosis. In xenograft model in vivo , shPrPc transfected cells were more sensitive to TRAIL-induced apoptosis than in shMock transfected cells. Molecular chemo-therapy approaches based on the regulation of PrPc expression need to address anti-tumor function of TRAIL under hypoxia. Molecular chemo-therapy approaches based on the regulation of PrPc expression need to address anti-tumor function of TRAIL under hypoxia.

Background Clustered regularly interspaced short palindromic repeats interference (CRISPRi) has provided an efficient approach for targeted gene inhibition. A non-model microorganism Halomonas species TD01 has been developed as a promising industrial producer of polyhydroxyalkanoates (PHA), a family of biodegradable polyesters accumulated by bacteria as a carbon and energy reserve compound. A controllable gene repression system, such as CRISPRi, is needed for Halomonas sp. TD01 to regulate its gene expression levels. Results For the first time CRISPRi was successfully used in Halomonas sp. TD01 to repress expression of ftsZ gene encoding bacterial fission ring formation protein, leading to an elongated cell morphology with typical filamentous shape similar to phenomenon observed with Escherichia coli. CRISPRi was employed to regulate expressions of prpC gene encoding 2-methylcitrate synthase for regulating 3-hydroxyvalerate monomer ratio in PHBV copolymers of 3-hydroxybutyrate (HB) and 3-hydroxyvalerate (HV). Percentages of HV in PHBV copolymers were controllable ranging from less than 1 to 13%. Furthermore, repressions on gltA gene encoding citrate synthase channeled more acetyl-CoA from the tricarboxylic acid (TCA) cycle to poly(3-hydroxybutyrate) (PHB) synthesis. The PHB accumulation by Halomonas sp. TD01 with its gltA gene repressed in various intensities via CRISPRi was increased by approximately 8% compared with the wild type control containing the CRISPRi vector without target. Conclusions It has now been confirmed that the CRISPRi system can be applied to Halomonas sp. TD01, a promising industrial strain for production of various PHA and chemicals under open and continuous fermentation process conditions. In details, the CRISPRi system was successfully designed in this study to target genes of ftsZ, prpC and gltA, achieving longer cell sizes, channeling more substrates to PHBV and PHB synthesis, respectively. CRISPRi can be expected to use for more metabolic engineering applications in non-model organisms. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0655-3) contains supplementary material, which is available to authorized users.

The cellular prion protein (PrPc) and hypoxia appear to be tightly intertwined. Beneficial effects of PrPc on neuronal survival under hypoxic conditions such as focal cerebral ischemia are strongly supported. Conversely, increasing evidence indicates detrimental effects of increased PrPc expression on cancer progression, another condition accompanied by low oxygen tensions. A switch between anaerobic and aerobic metabolism characterizes both conditions. A cellular process that might unite both is glycolysis. Putative role of PrPc in stimulation of glycolysis in times of need is indeed thought provoking. A significance of astrocytic PrPc expression for neuronal survival under hypoxic conditions and possible association of PrPc with the astrocyte-neuron lactate shuttle is considered. We posit PrPc-induced lactate production via transactivation of lactate dehydrogenase A by hypoxia inducible factor 1α as an important factor for survival of both neurons and tumor cells in hypoxic microenvironment. Concomitantly, we discuss a cross-talk between Wnt/β-catenin and PI3K/Akt signaling pathways in executing PrPc-induced activation of glycolysis. Finally, we would like to emphasize that we see a great potential in joining expertise from both fields, neuroscience and cancer research in revealing the mechanisms underlying hypoxia-related pathologies. PrPc may prove focal point for future research.

International audience; The cellular prion protein PrP(C)/CD230 is a GPI-anchor protein highly expressed in cells from the nervous and immune systems and well conserved among vertebrates. In the last decade, several studies suggested that PrP(C) displays antiviral properties by restricting the replication of different viruses, and in particular retroviruses such as murine leukemia virus (MuLV) and the human immunodeficiency virus type 1 (HIV-1). In this context, we previously showed that PrP(C) displays important similarities with the HIV-1 nucleocapsid protein and found that PrP(C) expression in a human cell line strongly reduced HIV-1 expression and virus production. Using different PrP(C) mutants, we report here that the anti-HIV-1 properties are mostly associated with the amino-terminal 24-KRPKP-28 basic domain. In agreement with its reported RNA chaperone activity, we found that PrP(C) binds to the viral genomic RNA of HIV-1 and negatively affects its translation. Using a combination of biochemical and cell imaging strategies, we found that PrP(C) colocalizes with the virus assembly machinery at the plasma membrane and at the virological synapse in infected T cells. Depletion of PrP(C) in infected T cells and microglial cells favors HIV-1 replication, confirming its negative impact on the HIV-1 life cycle.

A conformational conversion of the cellular prion protein (PrP(C)) is now recognized as the causal event of fatal neurodegenerative disorders, known as prion diseases. In spite of long-lasting efforts, however, the physiological role of PrP(C) remains unclear. It has been reported that PrP(C) is expressed in various areas of the olfactory system, including the olfactory epithelium, but its precise localization in olfactory sensory neurons (OSNs) is still debated. Here, using immunohistochemistry tools, we have reinvestigated the expression and localization of PrP(C) in the olfactory epithelium of adult congenic mice expressing different PrP(C) amounts, that is, wild-type, PrP-knockout, and transgenic PrP(C)-overexpressing animals. We found that PrP(C) was expressed in OSNs, in which, however, it was unevenly distributed, being detectable at low levels in cell bodies, dendrites and apical layer, and more abundantly in axons. We also studied the involvement of PrP(C) in the response of the olfactory epithelium to odorants, by comparing the electro-olfactograms of the 3 mouse lines subjected to different stimulation protocols. We found no significant difference between the 3 PrP genotypes, supporting previous reports that exclude a direct action of PrP(C) in the early signal transduction activity of the olfactory epithelium.

Spongiform degeneration is characterized by vacuolation in nervous tissue accompanied by neuronal death and gliosis. Although spongiform degeneration is a hallmark of prion diseases, this pathology is also present in the brains of patients suffering from Alzheimer’s disease, diffuse Lewy body disease, human immunodeficiency virus (HIV) infection, and Canavan’s spongiform leukodystrophy. The shared outcome of spongiform degeneration in these diverse diseases suggests that common cellular mechanisms must underlie the processes of spongiform change and neurodegeneration in the central nervous system. Immunohistochemical analysis of brain tissues reveals increased ubiquitin immunoreactivity in and around areas of spongiform change, suggesting the involvement of ubiquitin–proteasome system dysfunction in the pathogenesis of spongiform neurodegeneration. The link between aberrant ubiquitination and spongiform neurodegeneration has been strengthened by the discovery that a null mutation in the E3 ubiquitin–protein ligase mahogunin ring finger-1 (Mgrn1) causes an autosomal recessively inherited form of spongiform neurodegeneration in animals. Recent studies have begun to suggest that abnormal ubiquitination may alter intracellular signaling and cell functions via proteasome-dependent and proteasome-independent mechanisms, leading to spongiform degeneration and neuronal cell death. Further elucidation of the pathogenic pathways involved in spongiform neurodegeneration should facilitate the development of novel rational therapies for treating prion diseases, HIV infection, and other spongiform degenerative disorders.

Chantier qualité GA; A striking feature of the cellular prion protein (PrPC) is the heterogeneity of its glycoforms, whose contribution to PrPC function has yet to be defined. Using the 1C11 neuronal bioaminergic differentiation model and a glycomics approach, we show here a correlation between differential PrPC N-glycosylations in 1C115-HT serotonergic and 1C11NE noradrenergic cells compared to their 1C11 precursor cells and a variation of the glycogenome expression status in these cells. In particular, expression of genes involved in N-glycan synthesis or in the modeling of chondroitin and heparan sulfate proteoglycans appeared to be modulated. Our results highlight that, the expression of glycosylation-related genes is regulated during bioaminergic neuronal differentiation, consistent with a participation of glycoconjugates in neuronal development and plasticity. A neuronal regulation of glycosylation processes may have direct implications on some neurospecific functions of PrPC and may participate in specific brain targeting of prion strains.

Conversion of the cellular isoform of the prion protein (PrPC) into the disease-associated isoform (PrPSc) plays a key role in the development of prion diseases. Within its cellular pathway, PrPCundergoes several posttranslational modifications, i.e., the attachment of two N-linked glycans and a glycosyl phosphatidyl inositol (GPI) anchor, by which it is linked to the plasma membrane on the exterior cell surface. To study the interaction of PrPCwith model membranes, we purified posttranslationally modified PrPCfrom transgenic Chinese hamster ovary (CHO) cells. The mono-, di- and oligomeric states of PrPCfree in solution were analyzed by analytical ultracentrifugation. The interaction of PrPCwith model membranes was studied using both lipid vesicles in solution and lipid bilayers bound to a chip surface. The equilibrium and mechanism of PrPCassociation with the model membranes were analyzed by surface plasmon resonance. Depending on the degree of saturation of binding sites, the concentration of PrPCreleased from the membrane into aqueous solution was estimated at between 10-9and 10-7 M. This corresponds to a free energy of the insertion reaction of -48 kJ/mol. Consequences for the conversion of PrPCto PrPScare discussed.

The effects of heavy metals (Zn 2+ , Cu 2+ , and/or Cd 2+ ) on Escherichia coli expressing either prion (hPrP C ) or metallothionein 3 (MT-3) brain proteins capable of binding these metals were investigated. The expression of hPrP C or MT-3 in E.coli was confirmed using western-blot and dot-blot methods. After analyzing growth curves, we found that bacteria expressing prion protein better tolerated the presence of Zn 2+ in comparison with wild-type bacteria and bacteria expressing MT-3. The addition of Cd 2+ and Cu 2+ was well tolerated by bacteria expressing MT-3, whereas the bacteria expressing prion protein displayed slower growth when compared to the wild-type. We subsequently determined total content of the MT in bacteria using differential pulsed voltammetry (DPV), and depending on the treatment of the individual metals. MT expression in MT3 transformed cells as well as in control E.coli cells increased at the lowest metal concentration (25 µM), followed by a decrease at higher metal concentrations (50, 75, and 150 µM). The highest increase by Cd 2+ were observed. MT expression pattern in hPrP C transformed cells was different. After application of Cu 2+ an increase in MT expression continued also at higher metal concentrations; and after application of Cd 2+ and Zn 2+ no decrease in MT expression at higher metal concentrations was observed.

Prions are the etiological agent of fatal neurodegenerative diseases called prion diseases or transmissible spongiform encephalopathies. These maladies can be sporadic, genetic or infectious disorders. Prions are due to post-translational modifications of the cellular prion protein leading to the formation of a β-sheet enriched conformer with altered biochemical properties. The molecular events causing prion formation in sporadic prion diseases are still elusive. Recently, we published a research elucidating the contribution of major structural determinants and environmental factors in prion protein folding and stability. Our study highlighted the crucial role of octarepeats in stabilizing prion protein; the presence of a highly enthalpically stable intermediate state in prion-susceptible species; and the role of disulfide bridge in preserving native fold thus avoiding the misfolding to a β-sheet enriched isoform. Taking advantage from these findings, in this work we present new insights into structural determinants of prion protein folding and stability.

Purpose: Previous studies showed that lowering PrPC concomitantly reduced PrPSc in the brains of mice inoculated with prions. We aimed to develop assays that measure PrPC on the surface of human T98G glioblastoma and IMR32 neuroblastoma cells. Using these assays, we sought to identify chemical hits, confirmed hits, and scaffolds that potently lowered PrPC levels in human brains cells, without lethality, and that could achieve drug concentrations in the brain after oral or intraperitoneal dosing in mice. Methods: We utilized HTS ELISA assays to identify small molecules that lower PrPC levels by ≥30% on the cell surface of human glioblastoma (T98G) and neuroblastoma (IMR32) cells. Results: From 44,578 diverse chemical compounds tested, 138 hits were identified by single point confirmation (SPC) representing 7 chemical scaffolds in T98G cells, and 114 SPC hits representing 6 scaffolds found in IMR32 cells. When the confirmed SPC hits were combined with structurally related analogs, >300 compounds (representing 6 distinct chemical scaffolds) were tested for dose-response (EC50) in both cell lines, only studies in T98G cells identified compounds that reduced PrPC without killing the cells. EC50 values from 32 hits ranged from 65 nM to 4.1 μM. Twenty-eight were evaluated in vivo in pharmacokinetic studies after a single 10 mg/kg oral or intraperitoneal dose in mice. Our results showed brain concentrations as high as 16.2 μM, but only after intraperitoneal dosing. Conclusions: Our studies identified leads for future studies to determine which compounds might lower PrPC levels in rodent brain, and provide the basis of a therapeutic for fatal disorders caused by PrP prions. © 2014 Elsevier Ltd. All rights reserved.

Prion protein (PrPc) has been previously reported to be involved in gastric cancer (GC) development and progression. However, the association between expression of PrPc and GC prognosis is yet poorly characterized. In the present study, the expressions of PrPc and MGr1-Ag/37LRP, a protein interacting with PrPc, were detected using the tissue microarray technique and immunohistochemical method to compare clinicopathological parameters of 238 GC patients. We found that the expressions of PrPc and MGr1-Ag/37LRP were upregulated in GC lesions compared with their expressions in adjacent noncancerous tissues (p

Transmissible spongiform encephathalopathies or prion diseases are a group of neurological disorders characterized by neuronal loss, spongiform degeneration, and activation of astrocytes or microglia. These diseases affect humans and animals with an extremely high prevalence in some species such as deer and elk in North America. Although rare in humans, they result in a devastatingly swift neurological progression with dementia and ataxia. Patients usually die within a year of diagnosis. Prion diseases are familial, sporadic, iatrogenic, or transmissible. Human prion diseases include Kuru, sporadic, iatrogenic, and familial forms of Creutzfeldt–Jakob disease, variant Creutzfeldt–Jakob disease, Gerstmann–Sträussler–Scheinker disease, and fatal familial insomnia. The causative agent is a misfolded version of the physiological prion protein called PrPSc in the brain. There are a number of therapeutic options currently under investigation. A number of small molecules have had some success in delaying disease progression in animal models and mixed results in clinical trials, including pentosan polysulfate, quinacrine, and amphotericin B. More promisingly, immunotherapy has reported success in vitro and in vivo in animal studies and clinical trials. The three main branches of immunotherapy research are focus on antibody vaccines, dendritic cell vaccines, and adoptive transfer of physiological prion protein-specific CD4+ T-lymphocytes. Vaccines utilizing antibodies generally target disease-specific epitopes that are only exposed in the misfolded PrPSc conformation. Vaccines utilizing antigen-loaded dendritic cell have the ability to bypass immune tolerance and prime CD4+ cells to initiate an immune response. Adoptive transfer of CD4+ T-cells is another promising target as this cell type can orchestrate the adaptive immune response. Although more research into mechanisms and safety is required, these immunotherapies offer novel therapeutic targets for prion diseases., Video abstract

Transmissible spongiform encephalopathies, or prion diseases, are lethal neurodegenerative disorders caused by the infectious agent named prion, whose main constituent is an aberrant conformational isoform of the cellular prion protein, PrP(C) . The mechanisms of prion-associated neurodegeneration and the physiologic function of PrP(C) are still unclear, although it is now increasingly acknowledged that PrP(C) plays a role in cell differentiation and survival. PrP(C) thus exhibits dichotomic attributes, as it can switch from a benign function under normal conditions to the triggering of neuronal death during disease. By reviewing data from models of prion infection and PrP-knockout paradigms, here we discuss the possibility that Ca(2+) is the hidden factor behind the multifaceted behavior of PrP(C) . By featuring in almost all processes of cell signaling, Ca(2+) might explain diverse aspects of PrP(C) pathophysiology, including the recently proposed one in which PrP(C) acts as a mediator of synaptic degeneration in Alzheimer's disease.

Prionen verursachen transmissible spongiforme Enzephalopathien wie Scrapie, die bovine spongiforme Enzephalopathie oder die Creutzfeldt-Jakob-Krankheit und bestehen aus fehlgefaltetem, aggregierten Prionprotein (PrP). Aufgrund ihrer hohen Toleranz gegenüber üblichen Desinfektionsverfahren stellen sie sowohl eine besondere Herausforderung als auch ein exzellentes Paradigma für die Aufbereitung von chirurgischen Instrumenten dar. Tatsächlich haben sich Prionen bereits als aussagekräftige Modellpathogene für die Entwicklung neuer Formulierungen zur hochwirksamen Desinfektion auch von Bakterien, Viren und Pilzen bewährt. Um Prionen zukünftig in größerem Umfang als Testerreger zur Entwicklung neuer Mittel und Verfahren für die Breitspektrumdesinfektion ohne Rückgriff auf Tierversuche nutzen zu können, wurden in dieser Arbeit in vitro- Ersatzmethoden für Prion-Bioassays in Nagetieren aufgebaut und validiert. Zur quantitativen biochemischen Bestimmung der PrP-konvertierenden Keimaktivität des Scrapie-assoziierten Prionproteins wurde ein spezifisch adaptierter Protein Misfolding Cyclic Amplification (PMCA)-Assay etabliert. Mit hamster- adaptierten Scrapie-Prionen (Stamm 263K) kontaminierte Stahlstifte wurden verschiedenen Desinfektionsbehandlungen und anschließend einer quantitativen PMCA unterzogen. Die Ergebnisse des PMCA-Assays wurden dann mit bereits früher erhobenen Bioassay-Daten zur Reduktion infektiöser Priontiter durch die angewandten Desinfektionsverfahren verglichen. Zusätzlich dazu wurde ein Zellkulturassay mit primären Gliazellen aus Hamstern zum biologischen in vitro-Nachweis der Keimaktivität des Prionproteins entwickelt. Der Nachweis von Scrapie-assoziierter Keimaktivität an Stahlstiftprüfkörpern in der quantitativen PMCA übertraf die Sensitivität des Nachweises von Prioninfektiosität im Tierversuch um mindestens eine Größenordnung. Dabei konnte die Reduktion der Prioninfektiosität an Stahlstiftprüfkörpern durch 16 verschiedene Desinfektions-verfahren mit Titerreduktionen von weniger als 101-fach bis zu größer oder gleich 105,5-fach durchgängig korrekt abgeschätzt werden. Mittels PMCA biochemisch nachgewiesene Keimaktivität ließ sich auch biologisch in der Zellkultur detektieren. Konzeptionell deuten die Befunde dieser Arbeit darauf hin, dass die proteinöse Keimaktivität und pathogene Infektiosität von 263K Scrapie-Prionen biochemische und biologische Manifestationen desselben molekularen Prinzips darstellen. In praktischer Hinsicht steht mit der Gliazellkultur-gekoppelten PMCA potentiell eine gleichwertige oder sogar überlegene Alternative zum Prion-Bioassay in Tieren zur Verfügung., Prions are misfolded, aggregated prion proteins (PrP) and the causative agents of transmissible spongiform encephalopathies such as scrapie, bovine spongiform encephalopathy (BSE) or Creutzfeldt-Jakob disease. Prions are pathogens with an unusually high tolerance to inactivation and constitute a complex challenge as well as an informative paradigm for the re-processing of surgical instruments. They have been exploited successfully as model pathogens for the development of novel disinfectants with simultaneous activity also against bacteria, viruses and fungi. In order to further facilitate the use of prions as model agents in the search for novel broad-range disinfectants without the use of animal experiments we established and validated an experimental platform for the sensitive quantitative measurement and biological detection of scrapie seeding activity in vitro. We used specifically adapted serial protein misfolding cyclic amplification (PMCA) for the quantitative biochemical detection of the seeding activity of scrapie associated prion protein. Steel wires contaminated with 263K scrapie prions were subjected to different decontamination procedures and subsequently analysed by quantitative PMCA. The results of the PMCA assay were validated by comparison to bioassay data previously established for similar treatments of steel wires. Furthermore a novel cell culture assay of cerebral glial cell cultures from hamsters was established for the biological detection of 263K scrapie seeding activity. The detection limit for scrapie seeding activity on steel wires in the PMCA assay exceeded that for prion infectivity detected by bioassay at least tenfold. The residual scrapie infectivity’s on test wires treated with 16 different disinfection procedures yielding scrapie titre reductions of ≤101- to ≥105.5-fold could be correctly deduced, for all examined procedures, from our quantitative seeding activity assay. In addition, we found that scrapie seeding activity biochemically detected by PMCA could be also demonstrated biologically in the cell culture assay. Conceptually, the findings from this work suggested the seeding activity and infectivity of 263K scrapie prions as corresponding biochemical and biological manifestations of the same replicative molecular principle. In practical terms, our biochemical and biological in vitro assays, when combined, potentially provide a coequal or even superior alternative to titrations of scrapie infectivity in animals. In vitro-detection of the seeding activity of scrapie associated prion protein: A superior alternative to titrations of scrapie infectivity in animals

There are two central phenomena in prion disease: prion replication and prion neurotoxicity. Underlying them both is the conversion of a host-encoded ubiquitously expressed protein, prion protein (PrPC), into a partially-protease resistant isoform, PrPSc, which accumulates in the brain. PrPSc is associated with both pathology and infectivity. In the absence of PrPC, PrPSc cannot be generated and PrP-null mice do not propagate infectivity or develop pathology on infection with scrapie. However, while PrPC expression is fundamental to both prion infectivity and neurodegeneration, the uncoupling of these processes is a growing concept in the field. This dissociation is evident in subclinical states of prion infection, where PrPSc levels are high in the absence of disease, and in transgenic mice experiments, where, despite extra-neuronal PrPSc accumulation, even in very high amounts, there is no neurotoxicity. Both these models have further implications. Thus depleting PrPC from neurons (but not glia) of prion-infected mice prevents clinical disease, and detaching it from the surface of cells by removing its anchor does the same. The uncoupling toxicity and infectivity has implications for the nature of the neurotoxic species, these mouse models suggest that the site for the generation of this species is intra-neuronal. This review considers the role of neuronal surface-expressed PrPC in mediating toxicity in prion infection, and the dissociation of this from the deposition of PrPSc.

PrPC is highly expressed in both the central and peripheral nervous systems from early stages of development and in adulthood. Its major conformational change and conversion into an abnormal form (PrPSc) has been associated with the generation of prions, the infectious agent of transmissible spongiform encephalopathies (TSEs). The massive neurodegeneration presented by individuals suffering from these diseases has been associated with the gain of neurotoxic activity of PrPSc. On the other hand, major neurodegeneration is also observed in transgenic mice expressing PrPC molecules deleted of specific domains, which points to important functional domains within this molecule, and supports the hypothesis that loss-of PrPC function may contribute to the pathogenesis of TSEs. Furthermore, a large body of data demonstrates direct or indirect interaction of PrPC with extracellular matrix proteins, soluble factors, transmembrane proteins, G-protein coupled receptors and ions channels. The ability of PrPC to drive the assembly of multi-component complexes at the cell surface is likely the basis for its neurotrophic functions. These properties indicate that PrPC may be relevant for not only the spongiform encephalophaties, but also as an ancillary component of the pathogenesis of other neurodegenerative diseases, and therefore amenable to therapeutic targeting.

Metal induced free radicals are important mediators of neurotoxicity in several neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. Similar evidence is now emerging for prion diseases, a group of neurodegenerative disorders of humans and animals. The main pathogenic agent in all prion disorders is PrP-scrapie (PrP(Sc)), a beta-sheet rich isoform of a normal cell surface glycoprotein known as the prion protein (PrP(C)). Deposits of PrP(Sc) in the brain parenchyma are believed to induce neurotoxicity through poorly understood mechanisms. Recent reports suggest that imbalance of brain metal homeostasis is a significant cause of PrP(Sc)-associated neurotoxicity, though the underlying mechanisms are difficult to explain based on existing information. Proposed hypotheses include a functional role for PrP(C) in metal metabolism, and loss of this function due to aggregation to the disease associated PrP(Sc) form as the cause of brain metal imbalance. Other views suggest gain of toxic function by PrP(Sc) due to sequestration of PrP(C)-associated metals within the aggregates, resulting in the generation of redox-active PrP(Sc) complexes. The physiological implications of some PrP(C)-metal interactions are known, while others are still unclear. The pathological implications of PrP(C)-metal interaction include metal-induced oxidative damage, and in some instances conversion of PrP(C) to a PrP(Sc)-like form. Despite its significance, only limited information is available on PrP-metal interaction and its implications on prion disease pathogenesis. In this review, we summarize the physiological significance and pathological implications of PrP-metal interaction on prion disease pathogenesis.

While the prion protein (PrP) is clearly involved in neuropathology, its physiological roles remain elusive. Here, we demonstrate PrP functions in cell–substrate interaction in Drosophila S2, N2a and HeLa cells. PrP promotes cell spreading and/or filopodia formation when overexpressed, and lamellipodia when downregulated. Moreover, PrP normally accumulates in focal adhesions (FAs), and its downregulation leads to reduced FA numbers, increased FA length, along with Src and focal adhesion kinase (FAK) activation. Furthermore, its overexpression elicits the formation of novel FA-like structures, which require intact reggie/flotillin microdomains. Altogether, PrP modulates process formation and FA dynamics, possibly via signal transduction involving FAK and Src.

The prion protein is a cell surface glycoprotein whose physiological role remains elusive, while its implication in transmissible spongiform encephalopathies (TSEs) has been demonstrated. Multiple interactions between the prion protein and viruses have been described: viruses can act as co-factors in TSEs and life cycles of different viruses have been found to be controlled by prion modulation.We present data showing that human Adenovirus 5 induces prion expression. Inactivated Adenovirus did not alter prion transcription, while variants encoding for early products did, suggesting that the prion is stimulated by an early adenoviral function. Down-regulation of the prion through RNA interference showed that the prion controls adenovirus replication and expression.These data suggest that the prion protein could play a role in the defense strategy mounted by the host during viral infection, in a cell autonomous manner. These results have implications for the study of the prion protein and of associated TSEs.

Minutes Reasons for Non-Renewal 407.7.2 Double Representation

Approval of Minutes Election of Vice Chair Representation of Extension and RCDE on Faculty Senate 402.10.1 and 402.12.1(2)(3) Reasons for Non-Renewal 407.7.2

Approval of Minutes Representation of Extension and RCDE on Faculty Senate Academic Freedom and Professional Responsibility 403.1 and 403.3.1 Reasons for NonRenewal 407.7.2 Ad-hoc committee to review code Faculty Senate Supernumerary 402.3.1 Senate Standing Committees 402.12.1(2)(b) PRPC Vice Chair

EPC Curriculum Subcommittee Faculty Senate past president Faculty Forum EPC Membership FS President Supernumerary FSEC membership Cooperative extension

Faculty Forum membership Committee on Diversity and Equity EPC Curriculum Subcommittee EPC membership Faculty Senate past president

EPC Code Changes Faculty Forum Committee on Diversity and Equity Membership on EPC Committee

Modified code sections 405.7.2 and 405.8.3 Worked on code section 402.12.6 (5) Decided not to elect PRPC Vice Chair yet. Discussion on presence of an official copy of the code.

Discussion of December Faculty Senate meeting. PRPC charged to make changes to code requested by Committee on Committees. New Agriculture member needs to be appointed to PRPC. Dean's Tenure/Promotion Committee code added to FS agenda. Committee on Committees Discussion of clarifying/adding to code regarding termination dates. New chair elected.

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Proposals

Approved Minutes Report from March Faculty Senate Meeting Continuing Discussion New Business

Approved Minutes Report by PRPC chair on Faculty Senate meeting of February 6, 2006 Chair reported on items from previous PRPC meeting that will go forward to Senate Academic Rights and Responsibilities

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RK13 cell lines generated to express bovine PrPC with a four extra octarepeat insertional mutation (Bo-10ORPrPC) show partially insoluble PrPC and lower rates of cell growth when compared to either the same cells expressing wild type Bo-6ORPrPC or the original RK13 cell line. The expression of Bo-10ORPrPC in cell cultures was also associated with changes in cell size and reorganization of the actin cytoskeleton. This last process was reversed by Clostridium difficile toxin-B, a specific inhibitor of small GTPase proteins. Further, in clones expressing Bo-10ORPrPC, increased proportions of cells at cell cycle stage G2/M were observed. Proteasome inhibitors caused a further expansion of G2/M-stage cells that was more marked in cell lines expressing Bo-10ORPrPC than those expressing Bo-6ORPrPC, while this effect was minimal or null in the original RK13 cell line. Hence, the presence of Bo-10ORPrPC in RK13 cells promotes cell cycle arrest at G2/M, and the effect is amplified by proteasome inhibition. These findings suggest a role for PrPC in cell morphology and cell cycle regulation, and open new avenues for understanding the mechanisms underlying PrP mutation-associated diseases. © 2006 Federation of European Biochemical Studies.

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