Mouse neuronal CAD 5 cell line effectively propagates various strains of prions. Previously, we have shown that it can also be differentiated into the cells morphologically resembling neurons. Here, we demonstrate that CAD 5 cells chronically infected with prions undergo differentiation under the same conditions. To make our model more realistic, we triggered the differentiation in the 3D culture created by gentle rocking of CAD 5 cell suspension. Spheroids formed within 1 week and were fully developed in less than 3 weeks of culture. The mature spheroids had a median size of ~300 μm and could be cultured for up to 12 weeks. Increased expression of differentiation markers GAP 43, tyrosine hydroxylase, β-III-tubulin and SNAP 25 supported the differentiated status of the spheroid cells. The majority of them were found in the G0/G1 phase of the cell cycle, which is typical for differentiated cells. Moreover, half of the PrPC on the cell membrane was N-terminally truncated, similarly as in differentiated CAD 5 adherent cells. Finally, we demonstrated that spheroids could be created from prion-infected CAD 5 cells. The presence of prions was verified by immunohistochemistry, western blot and seed amplification assay. We also confirmed that the spheroids can be infected with the prions de novo. Our 3D culture model of differentiated CAD 5 cells is low cost, easy to produce and cultivable for weeks. We foresee its possible use in the testing of anti-prion compounds and future studies of prion formation dynamics.

Department of Pathology and Molecular Medicine 3rd Faculty of Medicine Charles University and Thomayer University Hospital Prague Czech Republic

Institute of Immunology and Microbiology 1st Faculty of Medicine Charles University Prague Czech Republic

Zobrazit více v PubMed

Ahn, M., Kalume, F., Pitstick, R., Oehler, A., Carlson, G., & DeArmond, S. J. (2016). Brain aggregates: An effective in vitro cell culture system modeling neurodegenerative diseases. Journal of Neuropathology and Experimental Neurology, 75, 256–262. https://doi.org/10.1093/jnen/nlv025

Alleaume‐Butaux, A., Dakowski, C., Pietri, M., Mouillet‐Richard, S., Launay, J. M., Kellermann, O., & Schneider, B. (2013). Cellular prion protein is required for neuritogenesis: Fine tuning of multiple signaling pathways involved in focal adhesions and actin cytoskeleton dynamics. Cell Health and Cytoskeleton, 5, 1–12.

Alleaume‐Butaux, A., Nicot, S., Pietri, M., Baudry, A., Dakowski, C., Tixador, P., Ardila‐Osorio, H., Haeberlé, A. M., Bailly, Y., Peyrin, J. M., Launay, J. M., Kellermann, O., & Schneider, B. (2015). Double‐edge sword of sustained ROCK activation in prion diseases through Neuritogenesis defects and prion accumulation. PLoS Pathogens, 11, e1005073. https://doi.org/10.1371/journal.ppat.1005073

Amin, L., Nguyen, X. T., Rolle, I. G., D'Este, E., Giachin, G., Tran, T. H., Šerbec, V., Cojoc, D., & Legname, G. (2016). Characterization of prion protein function by focal neurite stimulation. Journal of Cell Science, 129, 3878–3891. https://doi.org/10.1242/jcs.183137

Brandner, S., & Jaunmuktane, Z. (2017). Prion disease: Experimental models and reality. Acta Neuropathologica, 133, 197–222. https://doi.org/10.1007/s00401-017-1670-5

Caughey, B., Standke, H. G., Artikis, E., Hoyt, F., & Kraus, A. (2022). Pathogenic prion structures at high resolution. PLoS Pathogens, 18, e1010594. https://doi.org/10.1371/journal.ppat.1010594

Colby, D. W., & Prusiner, S. B. (2011). Prions (Vol. 3) (p. 3). Cold Spring Harb Perspect Biol. https://doi.org/10.1101/cshperspect.a006833

Collins, S. J., & Haigh, C. L. (2017). Simplified murine 3D neuronal cultures for investigating neuronal activity and neurodegeneration. Cell Biochemistry and Biophysics, 75, 3–13. https://doi.org/10.1007/s12013-016-0768-z

Didonna, A., Venturini, A. C., Hartman, K., Vranac, T., Curin Serbec, V., & Legname, G. (2015). Characterization of four new monoclonal antibodies against the distal N‐terminal region of PrP(c). PeerJ, 3, e811. https://doi.org/10.7717/peerj.811

Fang, C., Imberdis, T., Garza, M. C., Wille, H., & Harris, D. A. (2016). A neuronal culture system to detect prion synaptotoxicity. PLoS Pathogens, 12, e1005623. https://doi.org/10.1371/journal.ppat.1005623

Fremuntova, Z., Mosko, T., Soukup, J., Kucerova, J., Kostelanska, M., Hanusova, Z. B., Filipova, M., Cervenakova, L., & Holada, K. (2020). Changes in cellular prion protein expression, processing and localisation during differentiation of the neuronal cell line CAD 5. Biology of the Cell, 112, 1–21. https://doi.org/10.1111/boc.201900045

Galderisi, U., Jori, F. P., & Giordano, A. (2003). Cell cycle regulation and neural differentiation. Oncogene, 22, 5208–5219. https://doi.org/10.1038/sj.onc.1206558

Ghaemmaghami, S., Phuan, P. W., Perkins, B., Ullman, J., May, B. C., Cohen, F. E., & Prusiner, S. B. (2007). Cell division modulates prion accumulation in cultured cells. Proceedings of the National Academy of Sciences of the United States of America, 104, 17971–17976. https://doi.org/10.1073/pnas.0708372104

Giri, R. K., Young, R., Pitstick, R., DeArmond, S. J., Prusiner, S. B., & Carlson, G. A. (2006). Prion infection of mouse neurospheres. Proceedings of the National Academy of Sciences of the United States of America, 103, 3875–3880. https://doi.org/10.1073/pnas.0510902103

Glier, H., & Holada, K. (2012). Blood storage affects the detection of cellular prion protein on peripheral blood leukocytes and circulating dendritic cells in part by promoting platelet satellitism. Journal of Immunological Methods, 380, 65–72. https://doi.org/10.1016/j.jim.2012.04.002

González‐González, M. A., Gómez‐González, G. B., Becerra‐González, M., & Martínez‐Torres, A. (2017). Identification of novel cellular clusters define a specialized area in the cerebellar periventricular zone. Scientific Reports, 7, 40768. https://doi.org/10.1038/srep40768

Grassmann, A., Wolf, H., Hofmann, J., Graham, J., & Vorberg, I. (2013). Cellular aspects of prion replication in vitro. Viruses, 5, 374–405. https://doi.org/10.3390/v5010374

Harris, D. A., & True, H. L. (2006). New insights into prion structure and toxicity. Neuron, 50, 353–357. https://doi.org/10.1016/j.neuron.2006.04.020

He, S., Park, Y. H., Yorio, T., & Krishnamoorthy, R. R. (2015). Endothelin‐mediated changes in gene expression in isolated purified rat retinal ganglion cells. Investigative Ophthalmology & Visual Science, 56, 6144–6161. https://doi.org/10.1167/iovs.15-16569

Herbst, A., Aiken, J. M., & McKenzie, D. (2014). Replication of prions in differentiated muscle cells. Prion, 8, 166–168. https://doi.org/10.4161/pri.27890

Herva, M. E., Relano‐Gines, A., Villa, A., & Torres, J. M. (2010). Prion infection of differentiated neurospheres. Journal of Neuroscience Methods, 188, 270–275. https://doi.org/10.1016/j.jneumeth.2010.02.022

Hughes, D., & Halliday, M. (2017). What is our current understanding of PrP (Sc)‐associated neurotoxicity and its molecular underpinnings? Pathogens (Basel, Switzerland), 6, 6, 63. https://doi.org/10.3390/pathogens6040063

Iwamaru, Y., Takenouchi, T., Imamura, M., Shimizu, Y., Miyazawa, K., Mohri, S., Yokoyama, T., & Kitani, H. (2013). Prion replication elicits cytopathic changes in differentiated neurosphere cultures. Journal of Virology, 87, 8745–8755. https://doi.org/10.1128/JVI.00572-13

Jankovska, N., Rusina, R., Keller, J., Kukal, J., Bruzova, M., Parobkova, E., Olejar, T., & Matej, R. (2022). Biomarkers analysis and clinical manifestations in comorbid Creutzfeldt‐Jakob disease: A retrospective study in 215 autopsy cases. Biomedicine, 10(3), 680. https://doi.org/10.3390/biomedicines10030680

Janouskova, O., Rakusan, J., Karaskova, M., & Holada, K. (2012). Photodynamic inactivation of prions by disulfonated hydroxyaluminium phthalocyanine. The Journal of General Virology, 93, 2512–2517. https://doi.org/10.1099/vir.0.044727-0

Kim, B. H., Kim, J. I., Choi, E. K., Carp, R. I., & Kim, Y. S. (2005). A neuronal cell line that does not express either prion or doppel proteins. Neuroreport, 16, 425–429. https://doi.org/10.1097/00001756-200504040-00002

Kostelanska, M., & Holada, K. (2022). Prion strains differ in susceptibility to photodynamic oxidation. Molecules (Basel, Switzerland), 27, 27. https://doi.org/10.3390/molecules27030611

Kuwahara, C., Takeuchi, A. M., Nishimura, T., Haraguchi, K., Kubosaki, A., Matsumoto, Y., Saeki, K., Matsumoto, Y., Yokoyama, T., Itohara, S., & Onodera, T. (1999). Prions prevent neuronal cell‐line death. Nature, 400, 225–226. https://doi.org/10.1038/22241

Lancaster, M. A., Renner, M., Martin, C. A., Wenzel, D., Bicknell, L. S., Hurles, M. E., Homfray, T., Penninger, J. M., Jackson, A. P., & Knoblich, J. A. (2013). Cerebral organoids model human brain development and microcephaly. Nature, 501, 373–379. https://doi.org/10.1038/nature12517

Leary, E., Rhee, C., Wilks, B. T., & Morgan, J. R. (2018). Quantitative live‐cell confocal imaging of 3D spheroids in a high‐throughput format. SLAS Technology, 23, 231–242. https://doi.org/10.1177/2472630318756058

Lee, W. J., Chen, L. C., Lin, J. H., Cheng, T. C., Kuo, C. C., Wu, C. H., Chang, H. W., Tu, S. H., & Ho, Y. S. (2019). Melatonin promotes neuroblastoma cell differentiation by activating hyaluronan synthase 3‐induced mitophagy. Cancer Medicine, 8, 4821–4835. https://doi.org/10.1002/cam4.2389

Lee, Y. J., & Baskakov, I. V. (2013). The cellular form of the prion protein is involved in controlling cell cycle dynamics, self‐renewal, and the fate of human embryonic stem cell differentiation. Journal of Neurochemistry, 124, 310–322. https://doi.org/10.1111/j.1471-4159.2012.07913.x

Levine, D. J., Stöhr, J., Falese, L. E., Ollesch, J., Wille, H., Prusiner, S. B., & Long, J. R. (2015). Mechanism of scrapie prion precipitation with phosphotungstate anions. ACS Chemical Biology, 10, 1269–1277. https://doi.org/10.1021/cb5006239

Loubet, D., Dakowski, C., Pietri, M., Pradines, E., Bernard, S., Callebert, J., Ardila‐Osorio, H., Mouillet‐Richard, S., Launay, J. M., Kellermann, O., & Schneider, B. (2012). Neuritogenesis: The prion protein controls beta1 integrin signaling activity. FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology, 26, 678–690. https://doi.org/10.1096/fj.11-185579

Mahal, S. P., Baker, C. A., Demczyk, C. A., Smith, E. W., Julius, C., & Weissmann, C. (2007). Prion strain discrimination in cell culture: The cell panel assay. Proceedings of the National Academy of Sciences of the United States of America, 104, 20908–20913. https://doi.org/10.1073/pnas.0710054104

Martellucci, S., Santacroce, C., Santilli, F., Manganelli, V., Sorice, M., & Mattei, V. (2020). Prion protein in stem cells: A lipid raft component involved in the cellular differentiation process. International Journal of Molecular Sciences, 21, 4168. https://doi.org/10.3390/ijms21114168

Matej, R., Olejar, T., Janouskova, O., & Holada, K. (2012). Deletion of protease‐activated receptor 2 prolongs survival of scrapie‐inoculated mice. The Journal of General Virology, 93, 2057–2061. https://doi.org/10.1099/vir.0.043877-0

Moško, T., Galušková, S., Matěj, R., Brůžová, M., & Holada, K. (2021). Detection of prions in brain homogenates and CSF samples using a second‐generation RT‐QuIC assay: A useful tool for retrospective analysis of archived samples. Pathogens, 10(6), 750. https://doi.org/10.3390/pathogens10060750

Osterberg, N., & Roussa, E. (2009). Characterization of primary neurospheres generated from mouse ventral rostral hindbrain. Cell and Tissue Research, 336, 11–20. https://doi.org/10.1007/s00441-008-0743-0

Oz, S., Ivashko‐Pachima, Y., & Gozes, I. (2012). The ADNP derived peptide, NAP modulates the tubulin pool: Implication for neurotrophic and neuroprotective activities. PLoS ONE, 7, e51458. https://doi.org/10.1371/journal.pone.0051458

Panigaj, M., Glier, H., Wildova, M., & Holada, K. (2011). Expression of prion protein in mouse erythroid progenitors and differentiating murine erythroleukemia cells. PLoS ONE, 6, e24599. https://doi.org/10.1371/journal.pone.0024599

Pozarowski, P., & Darzynkiewicz, Z. (2004). Analysis of cell cycle by flow cytometry. Methods in Molecular Biology (Clifton, N.J.), 281, 301–311.

Prusiner, S. B. (1998). Prions. Proceedings of the National Academy of Sciences of the United States of America, 95, 13363–13383. https://doi.org/10.1073/pnas.95.23.13363

Qi, Y., Wang, J. K., McMillian, M., & Chikaraishi, D. M. (1997). Characterization of a CNS cell line, CAD, in which morphological differentiation is initiated by serum deprivation. The Journal of Neuroscience: the Official Journal of the Society for Neuroscience, 17, 1217–1225. https://doi.org/10.1523/JNEUROSCI.17-04-01217.1997

Reynolds, B. A., & Weiss, S. (1992). Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science (New York, N.Y.), 255, 1707–1710.

Seidel, D., Krinke, D., Jahnke, H. G., Hirche, A., Kloss, D., Mack, T. G., Striggow, F., & Robitzki, A. (2012). Induced tauopathy in a novel 3D‐culture model mediates neurodegenerative processes: A real‐time study on biochips. PLoS ONE, 7, e49150. https://doi.org/10.1371/journal.pone.0049150

Susaki, E. A., Tainaka, K., Perrin, D., Kishino, F., Tawara, T., Watanabe, T. M., Yokoyama, C., Onoe, H., Eguchi, M., Yamaguchi, S., Abe, T., Kiyonari, H., Shimizu, Y., Miyawaki, A., Yokota, H., & Ueda, H. R. (2014). Whole‐brain imaging with single‐cell resolution using chemical cocktails and computational analysis. Cell, 157, 726–739. https://doi.org/10.1016/j.cell.2014.03.042

Uchiyama, K., Tomita, M., Yano, M., Chida, J., Hara, H., Das, N. R., Nykjaer, A., & Sakaguchi, S. (2017). Prions amplify through degradation of the VPS10P sorting receptor sortilin. PLoS Pathogens, 13, e1006470. https://doi.org/10.1371/journal.ppat.1006470

Walters, R. O., & Haigh, C. L. (2023). Organoids for modeling prion diseases. Cell and Tissue Research, 392, 97–111. https://doi.org/10.1007/s00441-022-03589-x

Watts, J. C., Bourkas, M. E. C., & Arshad, H. (2018). The function of the cellular prion protein in health and disease. Acta Neuropathologica, 135, 159–178. https://doi.org/10.1007/s00401-017-1790-y

Wilham, J. M., Orrú, C. D., Bessen, R. A., Atarashi, R., Sano, K., Race, B., Meade‐White, K. D., Taubner, L. M., Timmes, A., & Caughey, B. (2010). Rapid end‐point quantitation of prion seeding activity with sensitivity comparable to bioassays. PLoS Pathogens, 6(12), e1001217. https://doi.org/10.1371/journal.ppat.1001217

Williamson, R. A., Peretz, D., Pinilla, C., Ball, H., Bastidas, R. B., Rozenshteyn, R., Houghten, R. A., Prusiner, S. B., & Burton, D. R. (1998). Mapping the prion protein using recombinant antibodies. Journal of Virology, 72, 9413–9418. https://doi.org/10.1128/JVI.72.11.9413-9418.1998

Yang, E., Liu, N., Tang, Y., Hu, Y., Zhang, P., Pan, C., Dong, S., Zhang, Y., & Tang, Z. (2015). Generation of neurospheres from human adipose‐derived stem cells. BioMed Research International, 2015, 743714. https://doi.org/10.1155/2015/743714

Yang, Z., & Wang, K. K. (2015). Glial fibrillary acidic protein: From intermediate filament assembly and gliosis to neurobiomarker. Trends in Neurosciences, 38, 364–374. https://doi.org/10.1016/j.tins.2015.04.003

Ye, X., Feng, T., Tammineni, P., Chang, Q., Jeong, Y. Y., Margolis, D. J., Cai, H., Kusnecov, A., & Cai, Q. (2017). Regulation of synaptic amyloid‐beta generation through BACE1 retrograde transport in a mouse model of Alzheimer's disease. The Journal of Neuroscience: the Official Journal of the Society for Neuroscience, 37, 2639–2655. https://doi.org/10.1523/JNEUROSCI.2851-16.2017

Zhang, Q., Zhao, Y. F., Xi, J. Y., Yu, W. B., & Xiao, B. G. (2016). Rho kinase II interference by small hairpin RNA ameliorates 1methyl4phenyl1,2,3,6tetrahydropyridineinduced parkinsonism in mice. Molecular Medicine Reports, 14, 4947–4956. https://doi.org/10.3892/mmr.2016.5889