Aiken, J

Amidian, S

Fernandez, E.V

Garza, M.C

Kamali-Jamil, R

Kim, C

McKenzie, D

Tancowny, B

Velasquez, C.D

Wang, X.Y

Wille, H.

Young, H

English

Repositorio Universidad de Zaragoza

138. Human cerebral organoids propagatesporadic CJD prionsBradley R. Grovemana, Simote Foliakia, Christina D.Orrua, Gianluigi Zanussob, James A. Carrolla, BrentRacea and Cathryn L. HaighaaLaboratory of Persistent Viral Diseases, National Institute of Allergy andInfectious Diseases, Division of Intramural Research, Rocky MountainLaboratories, National Institutes of Health, 903 South 4th Street,Hamilton, MT 59,840; bDepartment of Neurosciences, Biomedicine andMovement Sciences, University of Verona, Verona, ItalyCONTACT Cathryn L. Haigh cathryn.haigh@nih.govABSTRACTIntroduction: Human prion disease has traditionallybeen difficult to model in cell culture. Very fewhuman cell lines show sustained prion propagationover time and none represent three-dimensional (3D)brain tissue. Recently it was found that astrocyte cul-tures differentiated from human induced pluripotentstem cells (Hu-iPSCs) take up and propagate sporadicCJD (sCJD) prions with the resulting infection influ-enced by the sCJD subtype [1]. Hu-iPSCs have alsobeen utilized to differentiate self-patterning, structuredhuman brain tissues called cerebral organoids [2,3]. Wehypothesized that these cerebral organoids could beused to model human prion propagation in vitro.Materials and Methods: Cerebral organoid cultures weregenerated from hu-iPSCs using the protocol developed byLancaster and Knoblich [2]. Cerebral organoids wereinfected with one of two sCJD inocula and monitored forhealth, morphology, PrP seeding activity, PrP depositionand proteinase-k resistant PrP as compared with organoidsthat received a normal brain homogenate inoculum.Results: Cerebral organoids showed uptake and propa-gation of prions, detected by RT-QuIC analysis, follow-ing exposure to either sCJD inocula. Inoculum specificdifferences were observed. One inoculum showedrobust proteinase-K resistant PrP in all tested organoidsand coarse, granular PrP staining in the organoid inter-iors. The other inoculum induced a more toxic pheno-type that affected organoid health but displayed a lowlevel of prion seeding activity with no detectible pro-tease-resistant PrP.Conclusions: Cerebral organoid cultures can be used tomodel human prion disease in vitro. These structured 3Dmini-tissues offer a capacity to investigate aspects of priondiseases never previously available in a human neuronalcell model. Potential human disease-specific applicationsinclude delineating sCJD subtype pathogenesis and test-ing putative therapeutics.KEYWORDS: Cerebral organoid; sporadic CJD;induced pluripotent stem cell; RT-QuICReferences[1] Krejciova Z, Alibhai J, Zhao C, et al. Human stem cell-derived astrocytes replicate human prions in a PRNPgenotype-dependent manner. J Exp Med. 2017;214(12):3481–3495.[2] Lancaster MA and Knoblich JA. Generation of cerebralorganoids from human pluripotent stem cells. NatProtoc. 2014;9(10):2329–2340.[3] Renner M, Lancaster MA, Bian S, et al. Self-organizeddevelopmental patterning and differentiation in cere-bral organoids. Embo J. 2017;36(10):1316–1329139. Cryo-electron microscopy of chronic wastingdisease prionsSara Amidiana,b, Razieh Kamali-Jamila,b, Ester VázquezFernándeza,b, Maria Carmen Garzaa,b,$, Camilo DuqueVelásqueza,c, Xiongyao Wanga,b,#, Brian Tancownya,b,Chiye Kima, Judd Aikena,d, Debbie McKenziea,c,Howard Youngb and Holger Willea,baCentre for Prions and Protein Folding Diseases, University of Alberta,Edmonton, Canada; bDepartment of Biochemistry, University ofAlberta, Edmonton, Canada; cDepartment of Biological Sciences,University of Alberta, Edmonton, Canada; dDepartment of AnimalHealth and Biomedical Sciences, University of Alberta, Edmonton,CanadaCONTACT Sara Amidian samidian@ualberta.caPresent address: $University of Zaragoza, Zaragoza, Aragón, SpainPresent address: #School of Materials Science and Engineering,Harbin Institute of Technology, Weihai, Shandong, China.ABSTRACTIntroduction: Chronic wasting disease (CWD) is a fatalneurodegenerative disease in cervids that belongs to agroup of diseases known as prion diseases. CWD hasbeen detected in Canada, United States, South Korea,Norway, and most recently in Finland. CWD is the mostcontagious prion disease and poses a risk for transmissionto other species including humans.Aim: PrPSc is the misfolded form of the cellular prionprotein (PrPC) and the infectious agent that causes priondiseases. Themolecular mechanism of PrPC to PrPSc trans-formation is unknown. To understand this structural con-version, it is essential to understand the structure of PrPSc.Thus, the goal of this project is to investigate the structureof PrPSc forms that are causing CWD, using negative stainand cryo electron microscopy (EM).Method: PrPSc amyloid fibrilswere purified from the brainsof CWD-infected Tg33 mice, which express deer prion74 ABSTRACTprotein. To purify PrPSc amyloid fibrils, phosphotungstateanions and sarkosyl were used. In addition, enzymes suchas proteinase K (PK) or pronase E (PE) were added toremove PrPC. To assess the quality and quantity of thePrPSc fibrils, the purified amyloid fibrils were negativelystained and visualized using EM. Cryo-EM and 3D fibrilreconstruction are then performed on fibril preparations.Results and Conclusion: PrPSc amyloid fibrils weresuccessfully purified and visualized using negativestain EM. Both, PK- and PE-purified samples displayedcomplex yet similar morphologies. However, a domi-nant and recognizable type seen in both preparationsconsisted of filaments with a clear twisted ribbon-likemorphology. 2D class averages and 3D reconstructionsfrom the negatively stained micrographs could help tobetter classify these fibrils. Additionally, cryo-EM ana-lyses could resolve the structural differences betweenconformers of distinct CWD prion strains and theirfibrillization properties. PE-purified fibrils, on theother hand, exhibited striations that run perpendicularto the fibril axis. Interestingly, the distance betweenthese striations is ~40 nm, which corresponds to themolecular height of two PrPSc molecules in a four-rungbeta solenoid architecture that may adopt a head-to-head arrangement [1]. This observation could beexplained by the presence of sarkosyl molecules asso-ciating with the hydrophobic GPI-anchor of the stackedPrPSc molecules. Further image processing analyses willprovide a more complete understanding of the orienta-tion of the GPI-anchor in the CWD fibrils.References[1] Wille H, Requena J. The Structure of PrPSc Prions.Pathogens. 2018;7:20. DOI:10.3390/pathogens7010020.140. Molecular dynamics simulations of cervidprion protein variants to assess protein stabilityand susceptibility towards chronic wasting diseaseSara Amidiana,b, Lyudmyla Doroshc,d, Camilo DuqueVelásqueza,e, Maria Stepanovac,d, Judd Aikena,f,Debbie McKenziea,e and Holger Willea,baCentre for Prions and Protein Folding Diseases, University of Alberta,Edmonton, Canada; bDepartment of Biochemistry, University ofAlberta, Edmonton, Canada; cNational Institute for Nanotechnology,University of Alberta, Edmonton, Canada; dDepartment of Electricaland Computer Engineering, University of Alberta, Edmonton, Canada;eDepartment of Biological Sciences, University of Alberta, Edmonton,Canada; fDepartment of Animal Health and Biomedical Sciences,University of Alberta, Edmonton, Canada.CONTACT Sara Amidian samidian@ualberta.caABSTRACTIntroduction: Chronic wasting disease (CWD) is aprion disease that affects cervids. The central event inprion diseases is the structural conversion of thehealthy, cellular prion protein (PrPC) to the disease-causing isoform, PrPSc. It is technically difficult toexperimentally study the initial steps of the PrPC toPrPSc conversion; however, molecular dynamics (MD)simulation plays an important role in probing the mis-folding events. One approach is to assess the effect ofsingle-residue substitution on the structural stability ofPrPC. Polymorphisms in the prion protein gene caninfluence prion disease susceptibility, disease progres-sion, clinical presentation, and the propagation of dis-tinct CWD strains associated with different PrPScconformations. In this project, we use moleculardynamics simulation as a tool to characterize the effectof white-tailed deer (WTD) prion protein polymorph-isms Q95H and G96S on the experimentally deter-mined structure of the natively folded prion protein.Methods: The initial model for the WTD prion protein(residues 93–233) was generated using the crystal struc-ture of recombinant deer prion protein (PDB: 4YXH)as the template. The wild type (WT), 95H, and 96Smodels were subjected to minimizations, equilibrations,and production MD simulations using the Gromacspackage. In total, three independent 50ns simulationswere performed for each system.Results: The 96S polymorphism displayed a higher rootmean square deviation (RMSD) and root mean squarefluctuation (RMSF) values, which are indicators oflower structural stability compared to WT and 95Hforms. Also, 96S had a larger radius of gyration (Rg)values, indicating that the structure of 96S was lesscompactly folded than WT and 95H. Calculating thesolvent accessible surface area (SASA) of 96S revealedthat the structure of 96S was more exposed to thesolvent. On the other hand, structural dynamics of the95H polymorphism were closer to WT. RMSF and Rgvalues for 95H indicated higher stability and compact-ness of its structure. Additionally, the SASA values of95H were very similar to WT.Conclusions: Experimentally, the 96S allele is asso-ciated with slower disease progression and animalscarrying this polymorphism have longer incubationtime when inoculated with CWD prions. According toour MD results, it is apparent that the structure ofWTD prion protein carrying the 96S polymorphism isless stable than the WT protein. The clear difference inthe structural stability and local dynamics of 96SPRION 75

Cryo-electron microscopy of chronic wasting disease prions

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Cryo-electron microscopy of chronic wasting disease prions

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