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

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.

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.

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.