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Group I metabotropic glutamate receptors mediate slow excitatory neurotransmission in the central nervous system and are critical to activity-dependent synaptic plasticity, a cellular substrate of learning and memory. Dysregulated receptor signaling is implicated in neuropsychiatric conditions ranging from neurodevelopmental to neurodegenerative disorders. Importantly, group I metabotropic glutamate receptor signaling functions can be modulated by interacting proteins that mediate receptor trafficking, expression and coupling efficiency to signaling effectors. These interactions afford cell- or pathway-specific modulation to fine-tune receptor function, thus representing a potential target for pharmacological interventions in pathological conditions.

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.

Prion diseases, or transmissible spongiform encephalopathies (TSEs) are progressive, fatal neurodegenerative diseases with no effective treatment. The pathology of these diseases involves the conversion of a protease sensitive form of the cellular prion protein (PrPC) into a protease resistant infectious form (PrPres). The efficiency of this conversion is predicated upon a number of factors, most notably a strong homology between cellular PrPC and PrPres. In our recently published study, we infected mice with the RML-Chandler strain of scrapie and treated them with heterologous hamster prion proteins. This treatment was seen to reduce clinical signs of prion disease, to delay the onset of clinical symptoms and to prolong survival. In this current article we discuss potential mechanisms of action of treatment with heterologous prion proteins. We also discuss potential extensions of these studies using a heterologous rabbit PrP-based treatment strategy or a peptide based strategy, and improvement of treatment delivery including a lentiviral-based system.

Prion protein (PrPC) is connected with the origin of transmissive spongiform encephalopathies (TSEs), fatal diseases that are on the molecular level based on the conversion of the cellular form of prion protein, PrPC, into the infectious form, PrPTSE. This isoform, exhibiting increased resistance against proteases and common decontamination methods, accumulates in tissues and causes degenerative damages of the central nervous system. Potential physiological function of PrPC in cells remains unclear, though many efforts have been focused on this research area in past years. Expression of PrPC was detected especially in neurons, high levels of PrPC are also present in different types of cells of immune system. Whereas some immunocompetent cells were widely examined, the relationship of PrPC with the function of others was not studied. PrPC probably plays a role in differentiation and activation of some immune cells, participates in regulation of cytokine production and other immune processes, affects grow of CD4+ T-cell population and also takes a part in formation of secondary lymphatic organs. This bachelor thesis is focused on summarization of existing knowledge describing the role of the cellular prion protein in cells of immune system, which is important also from the point of view of diagnosis...

// 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.

Cancer is a group of diseases associated with abnormal cell growth, space occupation and metastasis. Tumours consist of a heterogeneous mix of cells including a small population of cells, called cancer stem cells (CSCs) that are able to self-renew and differentiate into all cell types of the tumour, and are proposed to drive tumourigenesis. Traditional chemotherapy kills most of differentiated cancer cells, but CSCs are inherently resistant and are able to reinitiate tumour growth. Accumulating evidence indicates that targeting both cancer cells and CSCs is required to elicit a cure. Therefore, it is a necessity to understand the biology of all the cells within a tumour. Cellular prion protein (PrPC) is an extracellular membrane glycoprotein that is mainly known for its association with transmissible spongiform encephalopathies. Several types of human cancer express high levels of PrPC including: breast, gastric, glioblastoma and colorectal. Notably, PrPC has roles in self-renewal, anti-apoptosis and differentiation. However, the role and proteolytic processing of PrPC in canine cancer cells and CSCs remain unclear. In this project, we aimed to investigate the expression levels and relative amounts of different isoforms of PrPC in a panel of different canine cancer cell lines; to study the proteolytic processing of PrPC in response to doxorubicin, a commonly used chemotherapy; and to develop and optimise an assay to determine the relative amount of secreted PrPC in cell culture medium. We have found that PrPC is detected in several canine cancer cell lines by western blot and that different cancer cell lines have different PRNP expression patterns. In response to DNA damage by doxorubicin, both a canine mammary inflammatory carcinoma (Lily) and a canine glioma (J3T) cell line, showed increases at the protein and the mRNA level of PrPC. PrPC is subject to proteolytic processes, including: α- cleavage, β-cleavage and shedding, which produce C1/N1, C2/N2 and full-length isoforms respectively. We have demonstrated that the ratio of full-length PrPC to C1 increases after DNA damage treatment. Conversely, the ratio of full-length PrPC to C1 decreased in CSCs. We also identified that different isoforms are degraded independently: the half-life of C1 becomes longer under the stress of chemotherapy, whereas, the half-life of full-length becomes shorter. Together these data indicate that the different isoforms may have independent biological significance in cancer cells. PrPC is also cleaved after the GPI anchor and secreted into the cell culture medium. To investigate the secreted isoforms of PrPC we developed and optimised an immunoprecipitation protocol. In conclusion, PrPC and its isoforms are detectable in different canine cancer cells and is expressed at different levels. The role of PrPC in cancer cells and CSCs may differ when stressed under chemotherapy, and we present evidence that C1 may act distinctively from full-length PrPC in response to chemotherapy. Future studies are needed to investigate the role of different isoforms of PrPC in canine cancer cells and CSCs in specific pathways relating to cell survival, apoptosis and metastatic potential, and to evaluate PrPC and its isoforms as a therapeutic target in oncotherapy.

The prion protein, PrPC, is a small, cell-surface glycoprotein notable primarily for its critical role in pathogenesis of the neurodegenerative disorders known as prion diseases. A hallmark of prion diseases is the conversion of PrPC into an abnormally folded isoform, which provides a template for further pathogenic conversion of PrPC, allowing disease to spread from cell to cell and, in some circumstances, to transfer to a new host. In addition to the putative neurotoxicity caused by the misfolded form(s), loss of normal PrPC function could be an integral part of the neurodegenerative processes and, consequently, significant research efforts have been directed toward determining the physiological functions of PrPC. In this review, we first summarise important aspects of the biochemistry of PrPC before moving on to address the current understanding of the various proposed functions of the protein, including details of the underlying molecular mechanisms potentially involved in these functions. Over years of study, PrPC has been associated with a wide array of different cellular processes and many interacting partners have been suggested. However, recent studies have cast doubt on the previously well-established links between PrPC and processes such as stress-protection, copper homeostasis and neuronal excitability. Instead, the functions best-supported by the current literature include regulation of myelin maintenance and of processes linked to cellular differentiation, including proliferation, adhesion, and control of cell morphology. Intriguing connections have also been made between PrPC and the modulation of circadian rhythm, glucose homeostasis, immune function and cellular iron uptake, all of which warrant further investigation.

International audience; Extracellular vesicles (EVs) are composed of microvesicles and exosomes. Exosomes are small membrane vesicles (40-120 nm sized) of endosomal origin released in the extracellular medium from cells when multivesicular bodies fuse with the plasma membrane, whereas microvesicles (i.e., shedding vesicles, 100 nm to 1 μm sized) bud from the plasma membrane. Exosomes and microvesicles carry functional proteins and nucleic acids (especially mRNAs and microRNAs) that can be transferred to surrounding cells and tissues and can impact multiple dimensions of the cellular life. Most of the cells, if not all, from neuronal to immune cells, release exosomes and microvesicles in the extracellular medium, and all biological fluids including blood (serum/plasma), urine, cerebrospinal fluid, and saliva contain EVs.Prion-infected cultured cells are known to secrete infectivity into their environment. We characterized this cell-free form of prions and showed that infectivity was associated with exosomes. Since exosomes are produced by a variety of cells, including cells that actively accumulate prions, they could be a vehicle for infectivity in body fluids and could participate to the dissemination of prions in the organism. In addition, such infectious exosomes also represent a natural, simple, biological material to get key information on the abnormal PrP forms associated with infectivity.In this chapter, we describe first a method that allows exosomes and microvesicles isolation from prion-infected cell cultures and in a second time the strategies to characterize the prions containing exosomes and their ability to disseminate the prion agent.

Prion diseases are a class of fatal neurodegenerative disorders that can be sporadic, genetic or iatrogenic. They are characterized by the unique nature of their etiologic agent: prions (PrPSc). A prion is an infectious protein with the ability to convert the host-encoded cellular prion protein (PrPC) into new prion molecules by acting as a template. Since Stanley B. Prusiner proposed the “protein-only” hypothesis for the first time, considerable effort has been put into defining the role played by PrPC in neurons. However, its physiological function remains unclear. This review summarizes the major findings that support the involvement of PrPC in signal transduction.

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.

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 1a as an important factor for survival of both neurons and tumor cells in hypoxic microenvironment. Concomitantly, we discuss a cross-talk between Wnt/b-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. Open-Access-Publikationsfonds 2016 peerReviewed

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.

The cellular prion protein (PrP(C)) is an ubiquitously expressed glycoprotein that is most abundant in the central nervous system. It is thought to play a role in many cellular processes, including neuroprotection, but may also contribute to Alzheimer's disease and some cancers. However, it is best known for its central role in the prion diseases, such as Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), and scrapie. These protein misfolding diseases can be sporadic, acquired, or genetic and are caused by refolding of endogenous PrP(C) into a beta sheet-rich, pathogenic form, PrP(Sc). Once prions are present in the central nervous system, they increase and spread during a long incubation period that is followed by a relatively short clinical disease phase, ending in death. PrP molecules can be broadly categorized as either 'good' (cellular) PrP(C) or 'bad' (scrapie prion-type) PrP(Sc), but both populations are heterogeneous and different forms of PrP(C) may influence various cellular activities. Both PrP(C) and PrP(Sc) are localized predominantly at the cell surface, with the C-terminus attached to the plasma membrane via a glycosyl-phosphatidylinositol (GPI) anchor and both can exist in cleaved forms. PrP(C) also has cytosolic and transmembrane forms, and PrP(Sc) is known to exist in a variety of conformations and aggregation states. Here, we discuss the roles of different PrP isoforms in sickness and in health, and show the subcellular distributions of several forms of PrP that are particularly relevant for PrP(C) to PrP(Sc) conversion and prion-induced pathology in the hippocampus. The Authors. Published by

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.

Mestrado em Biologia Molecular e Celular The present study was undertaken to identify proteins interacting with PrPC that could provide new insights into its physiological functions and pathological role. We performed a target search for lysosomal network protein, Rab7a and Rab9, in frontal cortex and cerebellum of human brain from patients with sCJD-MM1 and sCJD-VV2. The intracellular level of Rab7a was increased significantly, when compared with healthy age-matched control. Interactions of PrPC and Rab7a/Rab9 were further investigated by using confocal laser scanning microscopy. Immunofluorescence results suggested potential interactions of Rab7a and PrPC. siRNA against the Rab7a gene was used to knockdown the expression of Rab7a protein in primary cell culture of cortical neurons from wild type mice. This depleted Rab7a resulted an impairment of PrPC trafficking leading to an accumulation of PrPC in the endocytosis pathway. Furthermore, interactions of Tau and Rab7a were investigated by using western blot analysis and confocal laser scanning microscopy. Cell cultures of cortex of wildtype mice were treated with siRNA-Tau, siRNA-Rab7 and control siRNA followed by immunofluorescence. The results of immunofluorescence suggested potential interaction of Tau and Rab7a. Cells lines treated with siRNA-Tau, the intracellular levels of Rab7a and Rab9 significantly increases and their localization is also modified. When we transfected this cells lines with siRNA-rab7a the accumulation of Tau decreases in cytosolic region and their localization was also modified when compared with control cells. In conclusion, this study may help to understand and characterize the subtype specific disease progression in CJD cases. Furthermore, it could be a step ahead to development of new treatment strategies for diseases subtype specific manner. O presente estudo foi levado a cabo com o intuito de identificar possíveis proteínas que interajam com PrPC que possam fornecer novos conhecimentos sobre as suas funções fisiológicas assim como no papel patológico. O presente estudo incidiu na investigação dos níveis das proteínas endossomais Rab7a e Rab9 em amostras do córtex frontal e cerebelo de pacientes diagnosticados com sCJD-MM1 e sCJD-VV2. Observamos um aumento significativo dos níveis intracelulares do Rab7a em pacientes diagnosticados com sCJD-MM1 e sCJD-VV2 quando comparadas com amostras de pacientes na mesma faixa etária sem a doença (controlo). As interações entre PrPC e as proteínas Rab7a e Rab9 foram posteriormente estudadas com o auxílio à microscopia confocal. Os resultados da imunofluorescência sugeriram potenciais interações entre as proteínas Rab7a com o PrPC. O siRNA-Rab7 foi usado para diminuir a expressão da proteína Rab7a (“Knockdown”) na cultura primária de células do córtex de ratos saudáveis. Na cultura de células do córtex tratadas com o siRNA-Rab7, foi observado um emparelhamento da via endocítica. Seguidamente investigamos possíveis interações da proteína Tau com a proteína Rab7a, recorrendo ao western blot e a microscopia confocal. Culturas de células do córtex de ratos saudáveis foram tratadas com siRNA-Tau, siRNA-Rab7a e siRNA. Os resultados da imunofluorescência das diferentes culturas celulares sugeriram uma potencial interação entre as proteínas Tau e Rab7a. Nas linhas celulares tratadas com siRNA-Tau os níveis intracelulares das proteínas Rab7a e Rab9 aumentaram significativamente, assim como, foi também observada a alteração da sua localização. Nas células tratadas com siRNA-Rab7a observamos uma diminuição da acumulação da proteína Tau na região citosólica. Em conclusão, este trabalho pode ajudar a perceber e a caracterizar a progressão da doença nos subtipos específicos, no caso de sCJD. Contudo, este trabalho poderá ser também um passo à frente para o desenvolvimento de novas estratégias terapêuticas para os subtipos sCJD-MM1 e sCJD-VV2.

The cellular prion protein (PrP(C)), a cell surface glycoprotein involved in prion disorders, has been shown to mediate the toxicity of several pathological aggregates, including its own misfolded state and some oligomeric assemblies of the amyloid β peptide, which are thought to be primarily responsible for the synaptic dysfunction characterizing Alzheimer's disease. Thus, elucidating the physiological function of PrP(C), and how it could be corrupted by the interaction with misfolded proteins, may provide important insights to understand the pathological processes of prion and Alzheimer's diseases, and possibly other neurodegenerative disorders. In this manuscript, we review the data supporting a role for PrP(C) at the intersection of different neurodegenerative diseases, discuss potential mechanisms by which this protein could mediate neurotoxic signals, and examine therapeutic approaches that may arise from the identification of PrP(C)-directed compounds.

Prion proteins are infectious glycoproteins that do not contain nucleic acids. Their specific function in the brain is not yet fully understood. Conversion of the cellular form of prion protein (PrPC) into the pathological isoform (PrPSc) is regarded as the cause of neurodegenerative diseases. PrPC is a glycoprotein that interacts with many divalent metal ions, particularly Cu2+ and Zn2+. Another protein having the ability to bind metals, is also metallothionein (MT), which consists of several specific forms. Regarding formation of neurodegenerative diseases is significant metallothionein isoform MT-3 occuring in the brain. One of the MT-3 functions in the brain is its participation in maintaining of the optimal concentration of metal ions. The aim of this study was to verify the presence of expression PrPC or MT-3 genes in E. coli bacterial cells. Furthermore, we investigated the effect of metal additions on E. coli bacterial culture expressing PrPC or MT-3 protein. In the end we focused on determining the total concentration of MT in E. coli bacterial cells expressing the PrPC or MT-3 protein. Any determination was compared to a standard E. coli BL21 (DE3) strain.

The physiological role of the cellular prion protein (PrP(C)) is incompletely understood. The expression of PrP(C) in hematopoietic stem cells and immune cells suggests a role in the development of these cells, and in PrP(C) knockout animals altered immune cell proliferation and phagocytic function have been observed. Recently, a spontaneous nonsense mutation at codon 32 in the PRNP gene in goats of the Norwegian Dairy breed was discovered, rendering homozygous animals devoid of PrP(C). Here we report hematological and immunological analyses of homozygous goat kids lacking PrP(C) (PRNP(Ter/Ter) ) compared to heterozygous (PRNP (+/Ter)) and normal (PRNP (+/+)) kids. Levels of cell surface PrP(C) and PRNP mRNA in peripheral blood mononuclear cells (PBMCs) correlated well and were very low in PRNP (Ter/Ter), intermediate in PRNP (+/Ter) and high in PRNP (+/+) kids. The PRNP (Ter/Ter) animals had a shift in blood cell composition with an elevated number of red blood cells (RBCs) and a tendency toward a smaller mean RBC volume (P = 0.08) and an increased number of neutrophils (P = 0.068), all values within the reference ranges. Morphological investigations of blood smears and bone marrow imprints did not reveal irregularities. Studies of relative composition of PBMCs, phagocytic ability of monocytes and T-cell proliferation revealed no significant differences between the genotypes. Our data suggest that PrP(C) has a role in bone marrow physiology and warrant further studies of PrP(C) in erythroid and immune cell progenitors as well as differentiated effector cells also under stressful conditions. Altogether, this genetically unmanipulated PrP(C)-free animal model represents a unique opportunity to unveil the enigmatic physiology and function of PrP(C).