Small-Ruminant Lentivirus Enhances PrPSc Accumulation in Cultured Sheep Microglial Cells

ABSTRACT Sheep scrapie is the prototypical transmissible spongiform encephalopathy (prion disease), which has a fundamental pathogenesis involving conversion of normal cellular prion protein (PrPC [C superscript stands for cellular]) to disease-associated prion protein (PrPSc [Sc superscript stands for sheep scrapie]). Sheep microglial cell cultures, derived from a prnp 136VV/171QQ near-term fetal brain, were developed to study sheep scrapie in the natural host and to investigate potential cofactors in the prion conversion process. Two culture systems, a primary cell culture and a cell line transformed with the large T antigen of simian virus 40, were developed, and both were identified as microglial in origin as indicated by expression of several microglial phenotype markers. Following exposure to PrPSc, sheep microglial cells demonstrated relatively low levels (transformed cell line) to high levels (primary cell line) of PrPSc accumulation over time. The accumulated PrPSc demonstrated protease resistance, an inferred beta-sheet conformation (as determined by a commercial enzyme-linked immunosorbent assay), specific inhibition by anti-PrP antibodies, and was transmissible in a dose-dependent manner. Primary microglia coinfected with a small-ruminant lentivirus (caprine arthritis encephalitis virus-Cork strain) and PrPSc demonstrated an approximately twofold increase in PrPSc accumulation compared to that of primary microglia infected with PrPSc alone. The results demonstrate the in vitro utility of PrPSc-permissive sheep microglial cells in investigating the biology of natural prion diseases and show that small-ruminant lentiviruses enhance prion conversion in cultured sheep microglia.

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Propagation of a protease-resistant form of prion protein in long-term cultured human glioblastoma cell line T98G

Journal of General Virology ◽

10.1099/vir.0.80043-0 ◽

2004 ◽

Vol 85 (11) ◽

pp. 3449-3457 ◽

Cited By ~ 3

Author(s):

Yutaka Kikuchi ◽

Tomoshi Kakeya ◽

Ayako Sakai ◽

Kosuke Takatori ◽

Naoto Nakamura ◽

...

Keyword(s):

Cell Line ◽

Prion Protein ◽

Cellular Prion Protein ◽

Glioblastoma Cell Line ◽

Glioblastoma Cell ◽

Human Glioblastoma ◽

Human Glioblastoma Cell Line ◽

Human Glioblastoma Cell ◽

Resistant Form

Human prion diseases, such as Creutzfeldt–Jakob disease (CJD), a lethal, neurodegenerative condition, occur in sporadic, genetic and transmitted forms. CJD is associated with the conversion of normal cellular prion protein (PrPC) into a protease-resistant isoform (PrPres). The mechanism of the conversion has not been studied in human cell cultures, due to the lack of a model system. In this study, such a system has been developed by culturing cell lines. Human glioblastoma cell line T98G had no coding-region mutations of the prion protein gene, which was of the 129 M/V genotype, and expressed endogenous PrPC constitutively. T98G cells produced a form of proteinase K (PK)-resistant prion protein fragment following long-term culture and high passage number; its deglycosylated form was approximately 18 kDa. The PK-treated PrPres was detected by immunoblotting with the mAb 6H4, which recognizes residues 144–152, and a polyclonal anti-C-terminal antibody, but not by the mAb 3F4, which recognizes residues 109–112, or the anti-N-terminal mAb HUC2-13. These results suggest that PrPC was converted into a proteinase-resistant form of PrPres in T98G cells.

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The uptake of tau amyloid fibrils is facilitated by the cellular prion protein and hampers prion propagation in cultured cells

10.1101/2020.01.19.911644 ◽

2020 ◽

Author(s):

Elena De Cecco ◽

Luigi Celauro ◽

Silvia Vanni ◽

Micaela Grandolfo ◽

Adriano Aguzzi ◽

...

Keyword(s):

Prion Protein ◽

Amyloid Fibrils ◽

Cultured Cells ◽

Neuroblastoma Cells ◽

Cellular Prion Protein ◽

Brain Diseases ◽

Gene Encoding ◽

Prion Propagation ◽

Prion Conversion ◽

Tau Fibrils

AbstractTauopathies are prevalent, invariably fatal brain diseases for which no cure is available. Tauopathies progressively affect the brain through cell-to-cell transfer of tau protein amyloids, yet the spreading mechanisms are unknown. Here we show that the cellular prion protein (PrPC) facilitates the uptake of tau aggregates by cultured cells, possibly by acting as an endocytic receptor. In mouse neuroblastoma cells, we found that tau amyloids bind to PrPC; internalization of tau fibrils was reduced in isogenic cells devoid of the gene encoding PrPC. Antibodies against N-proximal epitopes of PrPC impaired the binding of tau amyloids and decreased their uptake. Surprisingly, exposure of chronically prion-infected cells to tau amyloids reduced the accumulation of aggregated prion protein; this effect lasted for more than 72 hours after amyloid removal. These results point to bidirectional interactions between the two proteins: whilst PrPC mediates the entrance of tau fibrils in cells, PrPSc buildup is greatly reduced in their presence, possibly because of an impairment in the prion conversion process.

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Species-specific anti-apoptotic activity of cellular prion protein in a mouse PrP-deficient neuronal cell line transfected with mouse, hamster, and bovine Prnp

Neuroscience Letters ◽

10.1016/j.neulet.2008.09.020 ◽

2008 ◽

Vol 446 (1) ◽

pp. 11-15 ◽

Cited By ~ 13

Author(s):

Guoying Wu ◽

Kenta Nakajima ◽

Natsumi Takeyama ◽

Masayoshi Yukawa ◽

Yojiro Taniuchi ◽

...

Keyword(s):

Cell Line ◽

Prion Protein ◽

Neuronal Cell ◽

Cellular Prion Protein ◽

Neuronal Cell Line ◽

Apoptotic Activity ◽

Species Specific

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Cellular Prion Protein and Caveolin-1 Interaction in a Neuronal Cell Line Precedes Fyn/Erk 1/2 Signal Transduction

Journal of Biomedicine and Biotechnology ◽

10.1155/jbb/2006/69469 ◽

2006 ◽

Vol 2006 ◽

pp. 1-13 ◽

Cited By ~ 28

Author(s):

Mattia Toni ◽

Enzo Spisni ◽

Cristiana Griffoni ◽

Spartaco Santi ◽

Massimo Riccio ◽

...

Keyword(s):

Signal Transduction ◽

Cell Line ◽

Prion Protein ◽

Neuronal Cell ◽

Cellular Prion Protein ◽

Glutathione S Transferase ◽

Neuronal Cell Line ◽

Caveolin 1 ◽

Fyn Kinase

It has been reported that cellular prion protein (PrPc) is enriched in caveolae or caveolae-like domains with caveolin-1 (Cav-1) participating to signal transduction events by Fyn kinase recruitment. By using the Glutathione-S-transferase (GST)-fusion proteins assay, we observed that PrPc strongly interacts in vitro with Cav-1. Thus, we ascertained the PrPc caveolar localization in a hypothalamic neuronal cell line (GN11), by confocal microscopy analysis, flotation on density gradient, and coimmunoprecipitation experiments. Following the anti-PrPc antibody-mediated stimulation of live GN11 cells, we observed that PrPc clustered on plasma membrane domains rich in Cav-1 in which Fyn kinase converged to be activated. After these events, a signaling cascade through p42/44 MAP kinase (Erk 1/2) was triggered, suggesting that following translocations from rafts to caveolae or caveolae-like domains PrPc could interact with Cav-1 and induce signal transduction events.

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Changes in cellular prion protein expression, processing and localisation during differentiation of the neuronal cell line CAD 5

Biology of the Cell ◽

10.1111/boc.201900045 ◽

2019 ◽

Vol 112 (1) ◽

pp. 1-21 ◽

Cited By ~ 3

Author(s):

Zuzana Fremuntova ◽

Tibor Mosko ◽

Jakub Soukup ◽

Johanka Kucerova ◽

Marie Kostelanska ◽

...

Keyword(s):

Protein Expression ◽

Cell Line ◽

Prion Protein ◽

Neuronal Cell ◽

Cellular Prion Protein ◽

Neuronal Cell Line ◽

Expression Processing

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Cellular prion protein suppresses the apoptosis in a neuronal cell line established from type-1 prion protein gene-deficient mice

Prions ◽

10.1007/4-431-29402-3_29 ◽

2006 ◽

pp. 203-203

Author(s):

Keiichi Saeki ◽

Takuya Nishimura ◽

Akikazu Sakudo ◽

Yoshitsugu Matsumoto ◽

Takashi Onodera

Keyword(s):

Cell Line ◽

Prion Protein ◽

Protein Gene ◽

Neuronal Cell ◽

Cellular Prion Protein ◽

Prion Protein Gene ◽

Neuronal Cell Line ◽

Deficient Mice

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Endogenous Brain Lipids Inhibit Prion Amyloid Formation In Vitro

Journal of Virology ◽

10.1128/jvi.02162-16 ◽

2017 ◽

Vol 91 (9) ◽

Cited By ~ 10

Author(s):

Clare E. Hoover ◽

Kristen A. Davenport ◽

Davin M. Henderson ◽

Mark D. Zabel ◽

Edward A. Hoover

Keyword(s):

Real Time ◽

Lipid Rafts ◽

Prion Protein ◽

Polar Lipid ◽

Cellular Prion Protein ◽

Conversion Process ◽

Amyloid Formation ◽

Inhibitory Function ◽

Prion Conversion

ABSTRACT The normal cellular prion protein (PrPC) resides in detergent-resistant outer membrane lipid rafts in which conversion to the pathogenic misfolded form is believed to occur. Once misfolding occurs, the pathogenic isoform polymerizes into highly stable amyloid fibrils. In vitro assays have demonstrated an intimate association between prion conversion and lipids, specifically phosphatidylethanolamine, which is a critical cofactor in the formation of synthetic infectious prions. In the current work, we demonstrate an alternative inhibitory function of lipids in the prion conversion process as assessed in vitro by real-time quaking-induced conversion (RT-QuIC). Using an alcohol-based extraction technique, we removed the lipid content from chronic wasting disease (CWD)-infected white-tailed deer brain homogenates and found that lipid extraction enabled RT-QuIC detection of CWD prions in a 2-log10-greater concentration of brain sample. Conversely, addition of brain-derived lipid extracts to CWD prion brain or lymph node samples inhibited amyloid formation in a dose-dependent manner. Subsequent lipid analysis demonstrated that this inhibitory function was restricted to the polar lipid fraction in brain. We further investigated three phospholipids commonly found in lipid membranes, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol, and found all three similarly inhibited RT-QuIC. These results demonstrating polar-lipid, and specifically phospholipid, inhibition of prion-seeded amyloid formation highlight the diverse roles lipid constituents may play in the prion conversion process. IMPORTANCE Prion conversion is likely influenced by lipid interactions, given the location of normal prion protein (PrPC) in lipid rafts and lipid cofactors generating infectious prions in in vitro models. Here, we use real-time quaking-induced conversion (RT-QuIC) to demonstrate that endogenous brain polar lipids can inhibit prion-seeded amyloid formation, suggesting that prion conversion is guided by an environment of proconversion and anticonversion lipids. These experiments also highlight the applicability of RT-QuIC to identify potential therapeutic inhibitors of prion conversion.

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Stability of BSE infectivity towards heat treatment even after proteolytic removal of prion protein

Veterinary Research ◽

10.1186/s13567-021-00928-8 ◽

2021 ◽

Vol 52 (1) ◽

Author(s):

Jan P. M. Langeveld ◽

Anne Balkema-Buschmann ◽

Dieter Becher ◽

Achim Thomzig ◽

Romolo Nonno ◽

...

Keyword(s):

Heat Treatment ◽

Prion Protein ◽

Bovine Brain ◽

Cellular Prion Protein ◽

Transmissible Spongiform Encephalopathies ◽

Spongiform Encephalopathies ◽

Similar Treatment ◽

Bank Voles ◽

Before And After ◽

Sheep Scrapie

AbstractThe unconventional infectious agents of transmissible spongiform encephalopathies (TSEs) are prions. Their infectivity co-appears with PrPSc, aberrant depositions of the host’s cellular prion protein (PrPC). Successive heat treatment in the presence of detergent and proteolysis by a keratinase from Bacillus licheniformis PWD-1 was shown before to destroy PrPSc from bovine TSE (BSE) and sheep scrapie diseased brain, however data regarding expected reduction of infectivity were still lacking. Therefore, transgenic Tgbov XV mice which are highly BSE susceptible were used to quantify infectivity before and after the bovine brain treatment procedure. Also four immunochemical analyses were applied to compare the levels of PrPSc. After heating at 115 °C with or without subsequent proteolysis, the original BSE infectivity of 106.2–6.4 ID50 g−1 was reduced to a remaining infectivity of 104.6–5.7 ID50 g−1 while strain characteristics were unaltered, even after precipitation with methanol. Surprisingly, PrPSc depletion was 5–800 times higher than the loss of infectivity. Similar treatment was applied on other prion strains, which were CWD1 in bank voles, 263 K scrapie in hamsters and sheep PG127 scrapie in tg338 ovinized mice. In these strains however, infectivity was already destroyed by heat only. These findings show the unusual heat resistance of BSE and support a role for an additional factor in prion formation as suggested elsewhere when producing prions from PrPC. Leftover material in the remaining PrPSc depleted BSE preparation offers a unique substrate for searching additional elements for prion infectivity and improving our concept about the nature of prions.

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Deciphering copper coordination in the animal prion protein amyloidogenic domain

10.1101/507624 ◽

2018 ◽

Author(s):

Giulia Salzano ◽

Martha Brennich ◽

Giordano Mancini ◽

Thanh Hoa Tran ◽

Giuseppe Legname ◽

...

Keyword(s):

Prion Protein ◽

Molecular Mechanisms ◽

Animal Species ◽

Cellular Prion Protein ◽

Transmissible Spongiform Encephalopathies ◽

Copper Binding ◽

Spongiform Encephalopathies ◽

X Ray ◽

Copper Coordination ◽

Prion Conversion

ABSTRACTPrions are pathological isoforms of the cellular prion protein (PrPC) responsible for transmissible spongiform encephalopathies (TSE). PrPC interacts with copper through unique octarepeat and non-octarepeat (non-OR) binding sites. Previous works on human PrPC suggest that copper binding to the non-OR region may have a role during prion conversion. The molecular details of copper coordination within the non-OR region are not well characterized. By means of small angle X-ray scattering (SAXS) and extended X-ray absorption fine structure (EXAFS) spectroscopy, we have investigated the Cu(II) structural effects on the protein folding and its coordination geometries when bound to the non-OR region of recombinant PrPC (recPrP) from animal species considered high or less resistant to TSE. As TSE-resistant model, we used ovine PrPC carrying the protective polymorphism at residues A136, R154 and R171 (OvPrP ARR); while as highly TSE-susceptible PrPC models we employed OvPrP with polymorphism V136, R154 and Q171 (OvPrP VRQ) and Bank vole recPrP (BvPrP). Our results reveal that Cu(II) affects the structural plasticity of the non-OR region leading to a more compacted conformation of recPrP. We also identified two Cu(II) coordinations in the non-OR region of these animal species. In type-1 coordination present in OvPrP ARR, Cu(II) is coordinated by four residues (S95, Q98, M109 and H111). Conversely, the type-2 coordination is present in OvPrP VRQ and BvPrP, where Cu(II) is coordinated by three residues (Q98, M109 and H111) and by one water molecule, making the non-OR region more flexible and open to the solvent. These changes in copper coordination in prion resistant and susceptible species provide new insights into the molecular mechanisms governing the resistance or susceptibility of certain species to TSE.

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