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15 Pág., 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., H. C. A. was supported by the CJD Foundation, Inc and Alzheimer Forschung Initiative e.V. (grant no.: 19050p); J. C. was supported by Spanish Ministry of Science award (grant no.: PID2021-122201OB-C21) cofunded by European Regional Development Fund. R. N. was supported by the Ministero della Salute (grant no.: RF-2016-02364498).

Background High-density oligomers of the prion protein (HDPs) have previously been identified in brain tissues of patients with rapidly progressive Alzheimer’s disease (rpAD). The current investigation aims at identifying interacting partners of HDPs in the rpAD brains to unravel the pathological involvement of HDPs in the rapid progression. Methods HDPs from the frontal cortex tissues of rpAD brains were isolated using sucrose density gradient centrifugation. Proteins interacting with HDPs were identified by co-immunoprecipitation coupled with mass spectrometry. Further verifications were carried out using proteomic tools, immunoblotting, and confocal laser scanning microscopy. Results We identified rpAD-specific HDP-interactors, including the growth arrest specific 2-like 2 protein (G2L2). Intriguingly, rpAD-specific disturbances were found in the localization of G2L2 and its associated proteins i.e., the end binding protein 1, α-tubulin, and β-actin. Discussion The results show the involvement of HDPs in the destabilization of the neuronal actin/tubulin infrastructure. We consider this disturbance to be a contributing factor for the rapid progression in rpAD.

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.

Cellular protein homeostasis (proteostasis) requires an accurate balance between protein biosynthesis, folding, and degradation, and its instability is causally related to human diseases and cancers. Here, we created numerous engineered cancer cell lines targeting APP (amyloid ß precursor protein) and/or PRNP (cellular prion) genes and we showed that APP knocking-down impaired PRNP mRNA level and vice versa, suggesting a link between their gene regulation. PRNP(KD), APP(KD) and PRNP(KD)/APP(KD) HeLa cells encountered major difficulties to grow in a 3D tissue-like environment. Unexpectedly, we found a cytoplasmic accumulation of the PrP(c) protein without PRNP gene up regulation, in both APP(KD) and APP(KO) HeLa cells. Interestingly, APP and/or PRNP gene ablation enhanced the chaperone/serine protease HTRA2 gene expression, which is a protein processing quality factor involved in Alzheimer's disease. Importantly, HTRA2 gene silencing decreased PRNP mRNA level and lowered PrP(c) protein amounts, and conversely, HTRA2 overexpression increased PRNP gene regulation and enhanced membrane-anchored and cytoplasmic PrP(c) fractions. PrP(c), APP and HTRA2 destabilized membrane-associated CD24 protein, suggesting changes in the lipid raft structure. Our data show for the first time that APP and the dual chaperone/serine protease HTRA2 protein could modulate PrP(c) proteostasis hampering cancer cell behavior.

Submitted by arlindo kohlrausch (ajfk@uepg.br) on 2022-03-03T13:44:25Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) _Mônica Garcia dos Santos.pdf: 1708542 bytes, checksum: 7663f8e3556d855bdf38b419748ce2f6 (MD5) Made available in DSpace on 2022-03-03T13:44:25Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) _Mônica Garcia dos Santos.pdf: 1708542 bytes, checksum: 7663f8e3556d855bdf38b419748ce2f6 (MD5) Previous issue date: 2021-12-17 O processo de diferenciação celular nos neurônios envolve a neuritogênese, que é a formação de neuritos. Os neuritos são projeções do corpo celular que se transformam em dendritos e axônios. Quando essa mudança ocorre, a célula promove as sinapses, intercomunicando-se com as outras ao seu redor. Nos estudos in vitro, existem protocolos de diferenciação que usam fatores neurotróficos e/ou ácido retinóico, combinados com soro fetal bovino (SFB) em concentrações menores das usadas para manutenção celular. Entretanto, o papel da redução do SFB ainda precisa ser definido. Neste estudo, o objetivo foi avaliar se apenas a redução de 10% para 1% ou 0% SFB poderia alterar a expressão de proteínas envolvidas na diferenciação neuronal, da mesma forma que os protocolos já estabelecidos alteram. A proteína sinaptofisina, presente na membrana de vesículas sinápticas, foi utilizada por ser um marcador neuronal clássico. Este estudo também analisou a proteína príon celular (PrPc ) e a proteína induzida por estresse 1 (STI1) como possíveis marcadores neuronais. Para isso, linhagens celulares de neuroblastoma humano (SH-SY5Y), neuroblastoma murino (Neuro2a) e feocromocitoma de rato (PC12) foram cultivadas em condições normais de manutenção com 10% SFB, em condições de redução de SFB a 1% e 0% e em condições de diferenciação usando ácido retinóico (AR) para os neuroblastomas e fator de crescimento de nervo (NGF) para feocromocitoma. Após os tempos de diferenciação, as células foram processadas para obtenção dos extratos, que foram analisados por western blot, usando os seguintes anticorpos: anti- sinaptofisina, antiPrPc , anti-STI1 e anti-actina. Nos experimentos com as células SH-SY5Y, a proteína sinaptofisina não foi detectada pelo anticorpo anti-sinaptofisina, indicando que não houve expressão desta proteína. Já a proteína PrPc apresentou resultados conflitantes, não sendo possível afirmar que sua expressão esteja envolvida na diferenciação. A linhagem Neuro2a apresentou maior expressão de sinaptofisina e de PrPc no grupo mantido a 1% SFB por 3 dias em relação ao grupo 10% SFB, tanto no ensaio de redução de SFB, como no ensaio de diferenciação com AR, mostrando-se independente de AR. No ensaio de redução de SFB, as células PC12 mantidas a 0% SFB apresentaram aumento na expressão das proteínas sinaptofisina e PrPc em relação ao grupo 10% SFB. No ensaio de diferenciação da linhagem PC12, foi possível observar o aumento de expressão de sinaptofisina nos grupos 1% SFB em relação ao grupo 10% SFB, mesmo sem NGF, e mostrou-se dependente de tempo. Porém, os grupos 1% SFB não apresentaram aumento na expressão de PrPc em relação ao grupo 10% SFB. Quanto à proteína STI1, não foi possível observar diferenças de expressão entre as condições, em nenhuma das linhagens. Conclui-se que, a redução de soro pode ser suficiente para indução da diferenciação nos estudos com PC12 mantidas a 0% SFB e em células Neuro2a nas condições de redução de SFB, mantidas a 1% SFB, utilizando como marcador neuronal sinaptofisina e, possivelmente, PrPc como novo marcador. Cell differentiation to neurons involves neuritogenesis, i. e., neurites formation. Neurites are protrusions of soma that become dendrites and axons. When this change occurs, cell promotes synapses, establishing neuronal connections. In vitro studies, there are differentiation protocols using neurotrophic factors and/or retinoic acid under fetal bovine serum (FBS) concentration reduction that differ normal condictions. However, the role of FBS reduction still needs to be defined. The aim of this study was to test whether only reduction of 10% to 1% or 0% FBS could alter proteins expression, likewise established protocols already alter. Synaptophysin, protein of synaptic vesicles, was used as classic neuronal marker. This study also analyzed cellular príon protein (PrPc ) and stress inducible protein 1 (STI1) as possible neuronal markers. For this, human neuroblastoma (SH-SY5Y), murine neuroblastoma (Neuro2a) and rat pheochromocytoma (PC12) cell lines were cultived under normal conditions with 10% FBS, FBS reduction conditions of 1% and 0% and differentiation conditions using retinoic acid (RA) for neuroblastomas and nerve growth fator (NGF) for pheochromocytoma. After differentiation time, cells were processed to obtain extracts, which were analized by western blot, using following antibodies: antisynaptophysin, anti-PrPc , anti-STI1 and anti-actin. In SH-SY5Y cells experiments, synaptophysin was not detected by antibody anti-synaptophisin, indicating there was no protein expression. PrPc protein presented conflicting results and it was not possible to say its involvement in differentiation. Neuro2a cell line presented higher expression of synaptophysin and PrPc in group maintained at 1% FBS for 3 days in relation to the 10% FBS group, both in FBS reduction assay, and in RA differentiation assay, showing to be independent of RA. In FBS reduction assay, PC12 cells maintained at 0% FBS showed an increase in the expression of synaptophysin and PrPc proteins in relation to 10% FBS group. In differentiation assay of PC12 cell line, it was possible to observe increase of synaptophysin expression in groups 1% FBS in relation to group 10% FBS, even without NGF, and it was shown to be time dependent. However, groups 1% FBS did not present an increase in PrPc expression in relation to group 10% FBS. Regarding STI1 protein, it was not possible to observe differences of expression between conditions, neither in cell lines. In conclusion, serum reduction can be sufficient to induce differentiation in studies with PC12 maintained to 0% FBS and in Neuro2a cells under FBS reduction conditions, maintained to 1% FBS, using synaptophysin as neural marker and possibly PrPc as a new marker.

Nelda A Rivera,1 Adam L Brandt,2 Jan E Novakofski,3 Nohra E Mateus-Pinilla1 1Illinois Natural History Survey-Prairie Research Institute, University of Illinois Urbana-Champaign, Champaign, IL, USA; 2Division of Natural Sciences, St. Norbert College, De Pere, WI, USA; 3Department of Animal Sciences, University of Illinois Urbana-Champaign, Urbana, IL, USACorrespondence: Nohra E Mateus-PinillaIllinois Natural History Survey-Prairie Research Institute, University of Illinois Urbana-Champaign, 1816 South Oak Street, Champaign, IL 61820, USATel +12173336856Fax +12172440802Email nohram@illinois.eduAbstract: Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) that affects members of the cervidae family. The infectious agent is a misfolded isoform (PrPSC) of the host prion protein (PrPC). The replication of PrPSC initiates a cascade of developmental changes that spread from cell to cell, individual to individual, and that for some TSEs, has crossed the species barrier. CWD can be transmitted horizontally and vertically, and it is the only TSE that affects free-ranging wildlife. While other TSEs are under control and even declining, infection rates of CWD continue to grow and the disease distribution continues to expand in North America and around the world. Since the first reported case in 1967, CWD has spread infecting captive and free-ranging cervids in 26 states in the US, 3 Canadian provinces, 3 European countries and has been found in captive cervids in South Korea. CWD causes considerable ecologic, economic and sociologic impact, as this is a 100% fatal highly contagious infectious disease, with no treatment or cure available. Because some TSEs have crossed the species barrier, the zoonotic potential of CWD is a concern for human health and continues to be investigated. Here we review the characteristics of the CWD prion protein, mechanisms of transmission and the role of genetics. We discuss the characteristics that contribute to prevalence and distribution. We also discuss the impact of CWD and review the management strategies that have been used to prevent and control the spread of CWD.Keywords: CWD, prion, PRNP, PrPC, PrPSC, TSE

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) that affects members of the cervidae family. The infectious agent is a misfolded isoform (PrPSC) of the host prion protein (PrPC). The replication of PrPSC initiates a cascade of developmental changes that spread from cell to cell, individual to individual, and that for some TSEs, has crossed the species barrier. CWD can be transmitted horizontally and vertically, and it is the only TSE that affects free-ranging wildlife. While other TSEs are under control and even declining, infection rates of CWD continue to grow and the disease distribution continues to expand in North America and around the world. Since the first reported case in 1967, CWD has spread infecting captive and free-ranging cervids in 26 states in the US, 3 Canadian provinces, 3 European countries and has been found in captive cervids in South Korea. CWD causes considerable ecologic, economic and sociologic impact, as this is a 100% fatal highly contagious infectious disease, with no treatment or cure available. Because some TSEs have crossed the species barrier, the zoonotic potential of CWD is a concern for human health and continues to be investigated. Here we review the characteristics of the CWD prion protein, mechanisms of transmission and the role of genetics. We discuss the characteristics that contribute to prevalence and distribution. We also discuss the impact of CWD and review the management strategies that have been used to prevent and control the spread of CWD.

The conversion of the normal cellular prion protein (PrPC) to the misfolded pathogenic scrapie prion protein (PrPSc) is the biochemical hallmark of prion replication. So far, various chemical compounds that inhibit this conformational conversion have been identified. Here, we report the novel anti-prion activity of SGI-1027 and its meta/meta analogue (M/M), previously known only as potent inhibitors of DNA methyltransferases (DNMTs). These compounds effectively decreased the level of PrPSc in cultured cells with permanent prion infection, without affecting PrPC at the transcriptional or translational levels. Furthermore, SGI-1027 prevented effective prion infection of the cells. In a PrP aggregation assay, both SGI-1027 and M/M blocked the formation of misfolded PrP aggregates, implying that binding of these compounds hinders the PrP conversion process. A series of binding and docking analyses demonstrated that both SGI-1027 and M/M directly interacted with the C-terminal globular domain of PrPC, but only SGI-1027 bound to a specific region of PrPC with high affinity, which correlates with its potent anti-prion efficacy. Therefore, we report SGI-1027 and related compounds as a novel class of potential anti-prion agents that preferentially function through direct interaction with PrPC., Graphical abstract A novel activity of DNMT inhibitor SGI-1027 was discovered to suppress the pathogenic process of prion. The direct binding of SGI-1027 to prion protein with a normal conformation was discovered to suppress the formation of misfolded prion proteins.Image 1

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 (*).

Anticancer drugs, such as fluorouracil (5-FU), oxaliplatin, and doxorubicin (Dox) are commonly used to treat colorectal cancer (CRC), however, owing to their low response rate and adverse effects, the development of efficient drug delivery systems (DDSs) is required. The cellular prion protein PrPC, which is a cell surface glycoprotein, has been demonstrated to be overexpressed in CRC, however, there has been no research on the development of PrPC-targeting DDSs for targeted drug delivery to CRC. In this study, PrPC aptamer (Apt)-conjugated gold nanoparticles (AuNPs) were synthesized for targeted delivery of Dox to CRC. Thiol-terminated PrPC-Apt was conjugated to AuNPs, followed by hybridization of its complementary DNA for drug loading. Finally, Dox was loaded onto the AuNPs to synthesize PrPC-Apt-functionalized doxorubicin-oligomer-AuNPs (PrPC-Apt DOA). The PrPC-Apt DOA were spherical nanoparticles with an average diameter of 20 nm. Treatment of CRC cells with PrPC-Apt DOA induced reactive oxygen species generation by decreasing catalase and superoxide dismutase activities. In addition, treatment with PrPC-Apt DOA inhibited mitochondrial functions by decreasing the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha, complex 4 activity, and oxygen consumption rates. Compared to free Dox, PrPC-Apt DOA decreased proliferation and increased apoptosis of CRC cells to a greater degree. In this study, we demonstrated that PrPC-Apt DOA targeting could effectively deliver Dox to CRC cells. PrPC-Apt DOA can be used as a treatment for CRC, and have the potential to replace existing anticancer drugs, such as 5-FU, oxaliplatin, and Dox.

Stanični prionski protein PrPC otkriven je u istraživanjima o uzroku prionopatija, koje karakterizira agregacija specifičnih proteina i posljedična neurodegeneracija. PrPC posjeduje dva mjesta za glikozilaciju i većina prionskog proteina izoliranog iz tkiva je diglikozilirana. Glikozilacija PrPC promovira transport PrPC do plazmatske membrane. S izuzetkom manje zastupljenih transmembranskih i cijepanih oblika, PrPC provodi veliku većinu životnog vijeka vezan za membranu putem GPI-sidra. Najveću količinu proteina PrPC nalazimo u imunosno privilegiranim mjestima, poput mozga, oka i placente te u imunosnom sustavu. Kako bi izbjegli nadzor imunosnog sustava domaćina, herpesvirusi su razvili raznolike mehanizme koji im omogućuju jedinstvenu sposobnost cjeloživotnog preživljavanja u inficiranim domaćinima u obliku latentne infekcije sa sposobnošću reaktivacije kad je imunosni sustav oslabljen. Dio mehanizama se temelji na sintezi proizvoda virusnih gena koji specifično zaustavljaju sintezu ili unutarstanični transport različitih efektorskih molekula imunosnog odgovora. Mišji citomegalovirus (MCMV), član obitelji obitelji herpesvirusa, dokazano mijenja reciklirajuće unutarstanične transportne puteve da bi smanjio ispoljavanje molekula bitnih za imunosnu reakciju na površini stanica. Svrha ovog rada bila je uspostaviti metodu za vizualizaciju i praćenje proteina PrPC ispoljenog na plazmatskoj membrani neuralnih stanica te uspostaviti metodu istovremenog praćenja četiri parametra u stanici putem konfokalne imunofluorescentne analize. Stanice neuroblastoma, N2A, bile su uzgojene, potom inficirane s virusom MCMV i zatim analizirane istovremeno na četiri parametara, putem četiri fluorofore, koje su bile marker za PrPC, jezgru, MCMV infekciju te stanični odjeljak, pri čemu je odabran AP1 pozitivan odjeljak. PrPC nije kolokalizirao s AP1 pozitvnim odjeljkom te vjerojatno MCMV ne utječe na njegov unutarstanični prijenos putem AP1 pozitivnog odjeljka., Cellular prion protein (PrPC) was discovered during research into the causes of prionopathis characterized by aggregation of specific proteins and subsequent neurodegeneration. PrPC possesses two glycosylation sites, and most tissue-isolated prion proteins are diglycosylated. PrPC glycosylation stimulates the transport of PrPC to the plasma membrane. With the exception of the less common transmembrane and split forms, PrPC spends most of its lifetime bound to a membrane with a GPI anchor. PrPC is most expressed in immunoprivileged sites, such as the brain, eye, and placenta, and in the immune system. To avoid controlling the host’s immune system, herpesviruses have developed various mechanisms that allow them to survive in infected hosts in the form of a latent infection with the ability to reactivate when the immune system is weakened. Several mechanisms are based on the synthesis of products of viral genes that specifically stop the synthesis or intracellular transport of various effector molecules of nonspecific and specific immune response. MCMV has been shown to alter the recycling of intracellular transport pathways to reduce the expression of molecules important for the immune response at the cell surface. The purpose of this study was to establish a method for visualization and monitoring of PrPC protein expressed on the plasma membrane of neural cells and to establish a method for simultaneous monitoring of four parameters in the cell by confocal immunofluorescence analysis. Neuroblastoma cells N2A were cultured, then infected with MCMV virus, and then analyzed simultaneously for four parameters, using 4 fluorophores, which were a marker for PrPC, nucleus, MCMV infection, and AP1. PrPC did not colocalize with the AP1 positive compartment and probably MCMV does not affect its intracellular sorting via the AP1 positive compartment.

Amyloid b peptide is cleaved from the amyloid precursor protein by b and γ secretases. According to the amyloid hypothesis i tis the main cause of the early pathogenetic events of Alzheimer's disease (AD) which is the most common neurodegenerative disease in the world without an effective treatment. The main pathogenesis of AD is considered to be the loss of synapses, disruption of neuronal plasticity and neurodegeneration. Amyloid b can bind directly to the membrane or mediate neuronal damage indirectly via toxic inflammatory mediators (e.g., reactive oxygen intermediates, nitric oxide and cytokines) by activating microglia and astrocytes. In addition to interacting with various membrane receptors, Ab can also bind to the cell surface directly, disrupting membrane integrity or forming selective cation channels. This thesis summarizes key interactions with membranes of synapses and mechanisms of amyloid- induced toxicity through receptors.

Studies on the cellular prion protein (PrPC) have been actively conducted because misfolded PrPC is known to cause transmissible spongiform encephalopathies or prion disease. PrPC is a glycophosphatidylinositol-anchored cell surface glycoprotein that has been reported to affect several cellular functions such as stress protection, cellular differentiation, mitochondrial homeostasis, circadian rhythm, myelin homeostasis, and immune modulation. Recently, it has also been reported that PrPC mediates tumor progression by enhancing the proliferation, metastasis, and drug resistance of cancer cells. In addition, PrPC regulates cancer stem cell properties by interacting with cancer stem cell marker proteins. In this review, we summarize how PrPC promotes tumor progression in terms of proliferation, metastasis, drug resistance, and cancer stem cell properties. In addition, we discuss strategies to treat tumors by modulating the function and expression of PrPC via the regulation of HSPA1L/HIF-1α expression and using an anti-prion antibody.

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.

The mobility of cellular prion protein (PrPC) in specific cell membrane domains and among distinct cell compartments dictates its molecular interactions and directs its cell function. PrPC works in concert with several partners to organize signaling platforms implicated in various cellular processes. The scaffold property of PrPC is able to gather a molecular repertoire to create heterogeneous membrane domains that favor endocytic events. Dynamic trafficking of PrPC through multiple pathways, in a well-orchestrated mechanism of intra and extracellular vesicular transport, defines its functional plasticity, and also assists the conversion and spreading of its infectious isoform associated with neurodegenerative diseases. In this review, we highlight how PrPC traffics across intra- and extracellular compartments and the consequences of this dynamic transport in governing cell functions and contributing to prion disease pathogenesis.

Cellular prion protein (PrP(C)) is a widely expressed glycosylphosphatidylinositol-anchored membrane protein. Scrapie prion protein is a misfolded and aggregated form of PrP(C) responsible for prion-induced neurodegenerative diseases. Understanding the function of the nonpathogenic PrP(C) monomer is an important objective. PrP(C) may be shed from the cell surface to generate soluble derivatives. Herein, we studied a recombinant derivative of PrP(C) (soluble cellular prion protein, S-PrP) that corresponds closely in sequence to a soluble form of PrP(C) shed from the cell surface by proteases in the A Disintegrin And Metalloprotease (ADAM) family. S-PrP activated cell-signaling in PC12 and N2a cells. TrkA was transactivated by Src family kinases and extracellular signal–regulated kinase 1/2 was activated downstream of Trk receptors. These cell-signaling events were dependent on the N-methyl-d-aspartate receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1), which functioned as a cell-signaling receptor system in lipid rafts. Membrane-anchored PrP(C) and neural cell adhesion molecule were not required for S-PrP–initiated cell-signaling. S-PrP promoted PC12 cell neurite outgrowth. This response required the NMDA-R, LRP1, Src family kinases, and Trk receptors. In Schwann cells, S-PrP interacted with the LRP1/NMDA-R system to activate extracellular signal–regulated kinase 1/2 and promote cell migration. The effects of S-PrP on PC12 cell neurite outgrowth and Schwann cell migration were similar to those caused by other proteins that engage the LRP1/NMDA-R system, including activated α(2)-macroglobulin and tissue-type plasminogen activator. Collectively, these results demonstrate that shed forms of PrP(C) may exhibit important biological activities in the central nervous system and the peripheral nervous system by serving as ligands for the LRP1/NMDA-R system.

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.

The vast majority of therapeutic approaches tested so far for prion diseases, transmissible neurodegenerative disorders of human and animals, tackled PrPSc , the aggregated and infectious isoform of the cellular prion protein (PrPC ), with largely unsuccessful results. Conversely, targeting PrPC expression, stability or cell surface localization are poorly explored strategies. We recently characterized the mode of action of chlorpromazine, an anti-psychotic drug known to inhibit prion replication and toxicity by inducing the re-localization of PrPC from the plasma membrane. Unfortunately, chlorpromazine possesses pharmacokinetic properties unsuitable for chronic use in vivo, namely low specificity and high toxicity. Here, we employed HEK293 cells stably expressing EGFP-PrP to carry out a semi-automated high content screening (HCS) of a chemical library directed at identifying non-cytotoxic molecules capable of specifically relocalizing PrPC from the plasma membrane as well as inhibiting prion replication in N2a cell cultures. We identified four candidate hits inducing a significant reduction in cell surface PrPC , one of which also inhibited prion propagation and toxicity in cell cultures in a strain-independent fashion. This study defines a new screening method and novel anti-prion compounds supporting the notion that removing PrPC from the cell surface could represent a viable therapeutic strategy for prion diseases.

The etiological agents’ nature has a pivotal role in the pathogenesis of infectious diseases. Prion diseases are unique in having a misfolded protein (PrPSc) as the sole transmissible agent. The host cellular protein (PrPC) is the substrate for misfolding and aggregation by PrPSc. Variability in the prion protein gene (PRNP) encoding PrPC affects prion disease progression and susceptibility, probably by inducing changes that may alter the efficiency of a pathogenic PrPC-to-PrPSc conversion. This principle applies to Chronic Wasting Disease (CWD; MONDO_0002680), a prion disease affecting members of the family Cervidae. CWD is widely distributed in North America from where it was imported to the Republic of Korea. Identification of CWD in Norway in wild reindeer (Rangifer tarandus) in 2016 and its subsequent identification in moose (Alces alces), and red deer (Cervus elaphus) has raised questions regarding the apparent geographical expansion of this disease in Europe. One key question targets the variation in the host PRNP, an important genetic risk factor in prion disease. This thesis studies PRNP variability in Norwegian cervids, where most European cases have been identified to June 2021. The endemic Norwegian species are reindeer, red deer, moose, and roe deer (Capreolus capreolus) with reindeer managed between, semi-domestic- or wild- populations. The first cases of CWD in Europe were recorded among the Norwegian wild reindeer population in Nordfjella 1, part of the Nordfjella management area in Southern Norway, where 19 CWD cases were identified. In this population, the risk to test positive to CWD was found to be greater in adult males and this likelihood increased with age. Similarly, two PRNP genotypes were found to increase the risk of CWD development in these wild reindeer. These susceptible genotypes combine two variants of PRNP (called alleles) named 226Q and deletion_226Q. Aware of the implications further spread of CWD could have for European cervid populations, a collaboration between scientists from Great Britain, the Czech Republic and Norway allowed to analyze the spatial variation of PRNP alleles across Europe. Using red deer as a proxy to monitor such variability, significant PRNP variation between countries and subregions was observed. The CWD susceptible allele 226Q was found in all European red deer subpopulations, although at variable frequencies. Spatial variation in PRNP alleles’ distribution was also observed within Norwegian populations of reindeer, red deer, and moose, but not roe deer, which was monomorphic. All species harbored the allele 226Q, but higher frequencies were found in reindeer, red deer, and moose in certain areas. The distribution of PRNP alleles differed between wild and semi-domesticated reindeer. Alleles associated with high susceptibility to CWD were identified in >55% of wild reindeer and 55 % av undersøkte villrein mot

Peripheral nerves transmit signals between peripheral tissue and the central nervous system, essential for normal motor and sensory function. To enable this, the integrity of both the axon and Schwann cell is crucial. The Schwann cell forms a myelin sheath around large diameter axons, necessary for rapid nerve conduction and axonal survival. Neuropathies, diseases affecting peripheral nerves, have diverse etiologies and may cause severe clinical signs in both human and animals. Some neuropathies are caused by inherited deficiencies in proteins necessary for normal axonal function or formation/maintenance of the myelin sheath. In this work, we have investigated how deficiencies in NDRG1 or PrPC affect peripheral nerve myelin maintenance by using morphological studies in two unique, spontaneous animal models. The hereditary polyneuropathy of Alaskan Malamutes is associated with a mutation in N-myc downstream-regulated gene 1 (NDRG1). Other mutations in the same gene cause polyneuropathies in humans, rodent models and Greyhound show dogs. However, the function of NDRG1 in peripheral nerves is largely unknown and the pathomorphological changes described in NDRG1-associated neuropathies of different species diverge. Whereas the human disease, denominated Charcot-Marie-Tooth disease type 4D, is considered a primary demyelinating disease with secondary axonal loss, axonal degeneration dominated in the polyneuropathy of Greyhounds. A detailed mapping of NDRG1 in canine tissues and cells presented in this thesis revealed high NDRG1 expression in epithelia and myelinating Schwann cells. A phosphorylated isoform of the protein localized to the centrosomes and cleavage furrow in a primary canine Schwann cell culture. Phosphorylated NDRG1 was restricted to the abaxonal cytoplasm of myelinating Schwann cells in control dogs, but this signal was not observed in an affected Alaskan Malamute. Ultrastructural studies and teased fiber preparations of nerves from neuropathic Alaskan Malamutes uncovered a demyelinating neuropathy associated with accumulation of actin in the Schwann cell and prominent myelin infoldings. The latter ultimately segregated the axon into pockets with aggregates of organelles, suggestive of early axonal degeneration. We therefore suggest that impaired NDRG1 function in the myelinating Schwann cells of affected Alaskan Malamutes results in deranged myelin maintenance, demyelination and secondary axonal degeneration. The cellular prion protein (PrPC) has been studied extensively because of its role in the development of transmissible spongiform encephalopathies in several species, such as Creutzfeldt-Jakobs disease in humans, bovine spongiform encephalopathy (“mad cow disease”) in cattle and scrapie in small ruminants. In these diseases, a misfolded isoform of the protein, PrPSc, accumulates in the brain and ultimately results in neuronal death. The normal functions of PrPC are not fully understood. Some of the phenotypes reported from transgenic mice lacking PrPC have later been ascribed to other genes, but recent reports have suggested that PrPC is important in dampening pro-inflammatory signaling and in myelin maintenance. We have used a nontransgenic, spontaneous model to investigate the latter; a line of Norwegian dairy goats lacking PrPC. Morphological studies revealed a demyelinating neuropathy with infiltration of macrophages and lymphocytes, vacuolated fibers and paranodal myelin outfoldings. Peripheral nerve lipid composition of adult goats lacking PrPC was significantly different from controls, while no difference was observed in young goats, suggesting a progressive disease. In conclusion, this is the first report of a demyelinating polyneuropathy caused by loss of PrPC in a nontransgenic mammal, supporting that PrPC has a vital role in peripheral nerve myelin maintenance. Perifere nerver overfører nerveimpulser mellom perifert vev og sentralnervesystemet, og er derfor essensielle for normal motorisk og sensorisk funksjon. For opprettholdelse av denne funksjonen, er både aksonet og den Schwannske cellen viktige. Aksonet er en utløper fra nervecellen og sørger for overføring av nerveimpulser. Den Schwannske cellen danner en myelinskjede rundt større aksoner. Dette er nødvendig for rask nerveledningshastighet og aksonal overlevelse. Nevropatier, sykdommer i perifere nerver, har ulik etiologi og kan forårsake alvorlige kliniske tegn både hos mennesker og dyr. Noen nevropatier er forårsaket av arvelige forstyrrelser i proteiner som er nødvendig for normal aksonal funksjon eller dannelse/vedlikehold av myelinskjeden. I dette arbeidet har vi undersøkt hvordan mangel på NDRG1 eller PrPC påvirker myelinvedlikehold i perifere nerver ved å bruke morfologiske studier i to unike, spontane dyremodeller. Arvelig polynevropati hos alaskan malamute er assosiert med en mutasjon i N-myc downstream-regulated gene 1 (NDRG1). Andre mutasjoner i samme genet forårsaker polynevropatier hos menneske, gnagermodeller og utstillingslinjer av greyhound. Funksjonen til NDRG1 i perifere nerver er imidlertid ukjent, og de patomorfologiske forandringene ved NDRG1-assosierte nevropatier hos ulike arter divergerer. Mens den humane nevropatien, Charcot-Marie-Tooths sykdom type 4D, er ansett for å være en primær demyeliniserende sykdom med sekundært aksonalt tap, dominerte aksonal degenerasjon ved polynevropati hos greyhound. En detaljert kartlegging av NDRG1 i celler og vev fra hund presentert i denne avhandlingen viser høye nivåer av NDRG1 i epitel og myeliniserende Schwannske celler. En fosforylert isoform av proteinet ble lokalisert i centrosomer og kløyvingsfuren hos Schwannske celler isolert fra hund. Fosforylert NDRG1 var begrenset til abaksonale cytoplasma i myeliniserende Schwannske celler i kontrollhunder, men dette signalet ble ikke observert i en syk alaskan malamute. Ultrastrukturelle studier og teased nerve fibers av nerver fra affiserte alaskan malamuter avslørte en demyeliniserende nevropati assosiert med aktinaggregater i de myeliniserende Schwannske cellene og uttalte innfoldinger av myelin. Innfoldingene delte stedvis aksonene inn i atskilte lommer med aggregater av organeller, forenelig med tidlig aksonal degenerasjon. Resultatene tyder på at svekket NDRG1-funksjon i myeliniserende Schwannske celler hos affiserte alaskan malamuter fører til forstyrret vedlikehold av myelin, demyelinisering og sekundær aksonal degenerasjon. Det cellulære prionproteinet (PrPC) har blitt grundig studert på grunn av sin rolle i utvikling av overførbare spongiforme encefalopatier, eller prionsykdommer, hos flere arter, for eksempel Creutzfeldt-Jakobs sykdom hos menneske, bovin spongiform encefalopati («kugalskap») hos storfe og skrapesjuke hos små drøvtyggere. Ved disse sykdommene vil en misfoldet isoform av proteinet, PrPSc, akkumulere i hjernen og tilslutt resultere i nevronal celledød. Normalfunksjonen til PrPC er ikke fullstendig forstått, og forståelsen av denne kan være viktig for å belyse den molekylære patogenesen ved prionsykdommer. Noen fenotyper rapportert fra transgene mus uten PrPC ble senere vist å skyldes andre gener, men nylige rapporter foreslår at PrPC er viktig for å dempe proinflammatorisk signalering og myelinvedlikehold. Vi har brukt en ny, ikke-transgen, spontan modell - en linje av norske melkegeiter som mangler PrPC - til å undersøke om denne modellen kan bekrefte tidligere studier fra transgene mus som har vist myelinskader. Morfologiske studier hos geiter uten PrPC avslørte en demyeliniserende nevropati med infiltrasjon av makrofager og lymfocytter, vakuoliserte nervefibre og paranodale myelinutfoldinger. Lipidsammensetningen i perifere nerver hos voksne geiter uten PrPC var signifikant forskjellig fra kontrollene, mens ingen forskjell ble observert hos unge dyr, noe som tyder på en progredierende sykdom. Dette er den første rapporten av en demyeliniserende polynevropati forårsaket av PrPC-mangel hos et ikke-transgent pattedyr, og støtter at PrPC har en viktig rolle i myelinvedlikehold.

Cellular prion protein (PrPC) is a plasma membrane glycophosphatidylinositol-anchored proteinand it is involved in multiple functions, including neuroprotection and oxidative stress. So far,most of the PrPC functional research is done in neuronal tissue or cell lines; the role of PrPC innon-neuronal tissues such as liver is only poorly understood. To characterize the role of PrPC inthe liver, a proteomics approach was applied in the liver tissue of PrPC knockout mice. Theproteome analysis and biochemical validations showed an excessive fat accumulation in the liverof PrPC knockout mice with a change in mRNA expression of genes linked to lipid metabolism. Inaddition, the higher Bax to Bcl2 ratio, up-regulation of tgfb1 mRNA expression in PrPC knockoutmice liver, further showed the evidences of metabolic disease. Over-expression of PrPC in fattyacid-treated AML12 hepatic cell line caused a reduction in excessive intracellular fat accumulation;shows association of PrPC levels and lipid metabolism. Therefore, based on observation ofexcessive fat globules in the liver of ageing PrPC knockout mice and the reduction of fataccumulation in AML12 cell line with PrPC over-expression, the role of PrPC in lipid metabolismis described. Open-Access-Publikationsfonds 2020 peerReviewed

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.

// Guanhong Luo 1, * , Weijie Wang 1, * , Qiong Wu 1, * , Yuanyuan Lu 1, * , Tao Su 2 , Nan Gu 3 , Kai Li 1 , Jingbo Wang 1 , Rui Du 4 , Xiaodi Zhao 1 , Xiaohua Li 1 , Rui Fan 1 , Hongbo Zhang 1 , Yongzhan Nie 1 , Xinmin Zhou 1 , Yongquan Shi 1 , Jie Liang 1 , Xin Wang 1 and Daiming Fan 1 1 State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, Xi'an, China 2 Department of Cardiology, Xijing Hospital, The Fourth Military Medical University, Xi'an, China 3 Department of Anesthesiology, Xijing Hospital, The Fourth Military Medical University, Xi'an, China 4 Department of Radiotherapy Oncology, Navy General Hospital, Beijing, China * These authors contributed equally to this work Correspondence to: Jie Liang, email: liangjie@fmmu.edu.cn Xin Wang, email: wangxinpaper@163.com Daiming Fan, email: fandaim@fmmu.edu.cn Keywords: PrP C , MGr1-Ag/37LRP, gastric cancer, multi-drug-resistance (MDR), apoptosis Received: August 02, 2016 Accepted: April 24, 2017 Published: May 11, 2017 ABSTRACT Cellular prion protein (PrP C ), the infective agent of transmissible spongiform encephalopathies, is thought to be related to several cellular physiological and physiopathological processes. We have previously reported that PrP C participates in multi-drug-resistance of gastric cancer. As the salient ligand molecule of PrP for participating in internalization and propagation of the scrapie form of prion protein (PrP Sc ), 37 kDa laminin receptor precursor protein (37LRP) shared the same gene coding sequence of MGr1-Ag, another protein previously found to be involved in multi-drug-resistance of gastric cancer in our lab. In the present study, we explored whether MGr1-Ag/37LRP contributed to PrP C mediated multi-drug-resistance in gastric cancer. Immunohistochemical staining showed similar expression patterns of MGr1-Ag/37LRP and PrPC in gastric cancer tissue serial sections. Western blot and immunohistochemistry also demonstrated correlative expression of MGr1-Ag/37LRP and PrP C in gastric cancer cell lines. Interaction between MGr1-Ag/37LRP and PrP C in gastric cancer cell lines and gastric cancer tissues were verified by immunofluorescence and co-immunoprecipitation. Furthermore, knockdown of MGr1-Ag/37LRP significantly attenuated PrP C induced multi-drug-resistance by sensitizing drug-induced apoptosis through inhibition of AKT activation. In conclusion, MGr1-Ag/37LRP may interact with PrP C and promote the PrP C induced multi-drug-resistance in gastric cancer through PI3K/AKT pathway. The current study elucidates the mechanism of how PrP C triggers intracellular signaling cascade resulting in multi-drug-resistance phenotype and provides a novel candidate molecular target against gastric cancer.

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.

Stanični prionski protein (engl. Prion Protein Cellular, PrPC) je sastavni dio različitih tkiva pri čemu je njegova fiziološka uloga i dalje predmet debate. Unutar stanice se može pronaći u više formi - citoplazmatskoj, transmembranskoj te formi koja je putem glikozilfosfatidilinozitolnog sidra pričvršćena na membranu, pri čemu je posljednja forma najraširenija. Pojedine forme proteina utvrđene su u gotovo svim odjeljcima stanice, što uključuje plazmatsku membranu, endoplazmatski retikulum, Golgijev aparat, endosome, pojedine vezikule te citoplazmu. Dodatnu kompleksnost u izučavanju biologije ovog proteina unose glikoforme, odnosno različite šećerne modifikacije, kojih ima nekoliko. Monoklonska protutijela, koja uspješno detektiraju različite molekule PrPC unutar stanice te omogućavaju praćenje pojedinih specifičnih (gliko)formi i određivanje njihova interaktoma, nezamjenjiv su alat u istraživanju biološke uloge PrPC. Ovim radom se utvrđivala djelotvornost novo-proizvedenih monoklonskih protutijela, u formi supernatanta, na sposobnost prepoznavanja mišjeg i humanog PrPC u metodama Western blot/imunoblot, imunofluorescentnoj mikroskopiji te protočnoj citometriji. Svi pokusi su rađeni na staničnim linijama mišjeg glioma GL261, mišjih embrionalnih fibroblasta MEF, mišjeg limfoma BW i transfektanti BW-PrP te na humanoj staničnoj liniji adenokarcinoma alveolarnih bazalnih epitelnih stanica A549, kao prikladnim staničnim modelima za ispitivanje PrPC i validacije monoklonskih protutijela na sposobnost prepoznavanja PrPC unutar stanice. Protutijela HuPrP.01, HuPrP.02 te HuPrP.04 su pokazala najveći potencijal u prepoznavanju humanog PrPC dok se u prepoznavanju mišjeg PrPC najboljim kandidatom pokazalo protutijelo MoPrP.03. Ova protutijela imaju potencijal za daljnji razvoj, koji će započeti proizvodnjom koncentriranih pripravaka poznate koncentracije protutijela putem pročišćavanja ispitanih supernatanata. Zanimljivo je kako je jedno od protutijela na humani PrPC, protutijelo 1G8, detektiralo PrPC putem imunofluorescentne mikroskopije u području membrane jezgre, gdje isti protein još do sada nije opisan. Zaključno, na temelju ukupnih rezultata prikupljenih različitim metodama, utvrdili smo koja novo-proizvedena protutijela vrijedi dalje razvijati, pročišćavati i validirati te koristiti u budućim istraživanjima bioloških uloga PrPC., PrPC is an integral part of different tissues, and its physiological role remains the subject of debate. Within the cell, this protein can be found in several forms, including a cytoplasmic form and a transmembrane form. However, the most widespread form is one that is attached to the membrane through a glycosylphosphatidylinositol anchor. Certain protein forms have been found in almost all cell compartments including the plasma membrane, endoplasmic reticulum, Golgi apparatus, endosomes and other vesicles, as well as the cytoplasm. An additional level of complexity in the study of the biology of this protein is its glycoprofile, or different sugar modifications, of which there are several. Monoclonal antibodies that successfully detect different PrPC molecules within cells also enable the monitoring of specific forms of PrPC (glyco)forms and determination of the PrPC interactome. The present study determined the efficacy of newly-produced monoclonal antibodies to recognize mouse and human PrPC in Western blotting/immunoblotting, immunofluorescence microscopy, and flow cytometry. Antibodies HuPrP.01, HuPrP.02 and HuPrP.04 showed the greatest potential for recognition of human PrPC, while the antibody MoPrP.03 showed best capability in recognition of mouse PrPC. These antibodies have the potential for further development, which will start by the production of concentrated preparations of known antibody concentrations by purifying the supernatants that were tested. Interestingly, one of the anti-HuPrP antibodies, 1G8, detected immunofluorescence signal around the nucleus membrane, where the PrPC localization has not been previously described. Based on the obtained results, we have determined which newly-produced antibodies are candidates for further development, purification and validation, and can be used in future research of the biological role of PrPC.

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.

Transmissible spongiform encephalopathies (TSEs) are rare, uniformly fatal neurodegenerative disorders that can affect many mammalian species, including humans. A hallmark of these diseases is the conversion of cellular prion protein (PrPC) into an abnormally folded form. This misfolded PrPC is infectious, since it can provide a template for pathogenic conversion of PrPC in a new host. In addition to any toxicity of the misfolded protein, loss of normal PrPC function could be involved in the neurodegenerative processes. However, the physiological role of PrPC is still poorly understood and this project has aimed to address that lack of knowledge. Out of the many putative functions ascribed to PrPC, the most commonly proposed is that it protects cells from stress. In contrast, I have found that stable transfection of the prion protein gene into SH-SY5Y neuroblastoma cells increases cell death in response to serum removal from the culture medium. Following treatment with several chemical toxins, two out of four stably transfected clones did, generally, display greater viability than untransfected cells that do not express detectable levels of PrPC. However, knockdown of PrPC expression by RNA interference had no effect on this stress resistance, indicating that it may not have been mediated directly by PrPC. Given the lack of robust stress protection afforded by PrPC transfection, proteomic analyses of the cells were carried out to identify alternative processes that were perturbed as a result of PrPC expression. The results obtained suggested roles for PrPC in cytoskeletal organisation and cell cycle regulation. Various proteins involved in cytoskeletal organisation were confirmed by western blotting to be differentially expressed in some or all of the stably transfected clones. Additionally, the expression changes to proteins involved in cell cycle regulation resulted in slower proliferation of the clones compared with untransfected cells, a difference that was reduced following RNA interference-mediated knockdown of PrPC. Taken together, these data suggested that specific growth factor-activated pathways were differentially regulated in the stably transfected clones. One candidate pathway was nerve growth factor (NGF) signalling, which promotes neuronal survival and differentiation as well as regulating various processes outside of the nervous system. PrPC-transfection resulted in altered expression of receptors for NGF, suggesting that the stably transfected clones were, indeed, responding differently to NGF stimulation. However, the molecular mechanism responsible for these expression changes remains to be determined, since co-immunoprecipitation experiments did not identify any physical interactions between PrPC and the NGF receptors. Nonetheless, a role for PrPC in modulating NGF signalling has the potential to explain many of the diverse phenotypic observations in PrPC-null mice and might indicate that loss of PrPC function is an important part of TSE pathogenesis.

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 family of transmissible, progressive, and uniformly fatal neurodegenerative disorders that affect humans and animals. Although cross-species transmissions of prions are usually limited by an apparent “species barrier”, the spread of a prion disease to humans by ingestion of contaminated food, or via other routes of exposure, indicates that animal prions can pose a significant public health risk. The infectious agent responsible for the transmission of prion diseases is a misfolded conformer of the prion protein, PrP Sc , a pathogenic isoform of the host-encoded, cellular prion protein, PrP C . The detailed mechanisms of prion conversion and replication, as well as the high-resolution structure of PrP Sc , are unknown. This review will discuss the general background related to prion biology and assess the structural models proposed to date, while highlighting the experimental challenges of elucidating the structure of PrP Sc . Keywords: prion; cellular prion protein; PrP

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.