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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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Biology

Undersøgelse Spredningen og toksicitet af Prion-lignende proteiner Brug af metazoan modelorganismen<em> C. elegans</em

Published: January 08, 2015
doi:
10.3791/52321
, , , ,
1Department of Molecular Biosciences, Rice Institute for Biomedical Research,Northwestern University

Summary

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

Abstract

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Introduction

Mange neurodegenerative sygdomme, herunder Alzheimers sygdom (AD), Parkinsons sygdom (PD), amyotrofisk lateral sklerose (ALS), og overførbare spongiforme encephalopatier (TSE), er forbundet med sammenlægning-tilbøjelige proteiner og er derfor kollektivt kendt som protein misfoldning lidelser (PMDs ). TSE eller prionsygdomme udgør en unik klasse af PMDs i, at de kan være infektiøs i både mennesker og dyr 1. På molekylært niveau, prioner replikere ved at rekruttere og omdannelse monomert α-helix-rige vært kodet cellulær PrP (PrP C) i den patologiske β-ark-rige PrP Sc konformation 2,3. Selvpropagerende proteinaggregater er også identificeret i svampe, som deler vigtige egenskaber med pattedyr prioner 4,5. Derudover mammale prioner er i stand til at bevæge sig fra celle til celle og inficere naive celler 6,7.

Mens PMDs othER end TSE er ikke smitsomme, de deler en fælles patogen princip med prionsygdomme 8,9. Selvom proteiner knyttet til hver af de PMDs ikke er forbundet i struktur eller funktion, de alle danne aggregater via en krystallisation-lignende proces kaldet nukleeret eller podet polymerisation; desuden proteinholdige frø vokse ved at ansætte deres opløselige isoformer 2,10,11. Effektiviteten til selv Propagate varierer in vivo, afhængig af de iboende egenskaber af proteinet, som sammen med yderligere cellulære faktorer såsom molekylære chaperoner i sidste ende bestemmer satser samlede kernedannelse, såning, fragmentering og spredning 12-15. Derfor skal der findes en fin balance mellem disse faktorer, der muliggør effektiv udbredelse af proteinaggregering. Dette kan også forklare, hvorfor kun nogle amyloidogene aggregater harbor kendetegnene for en prion, og dermed ikke alle PMDs er infektiøse. Prioner synes at repræsentere 'bedste lande' ofa bredt spektrum af selv-replikerende proteinholdige aggregater, hvilket gør dem et kraftfuldt værktøj til at studere PMDs 8,13.

Interessant nok toksiciteten forbundet med sygdomsrelaterede aggregater ofte har en ikke celle autonom komponent 16,17. Dette betyder, at de påvirker tilstødende celler, som ikke udtrykker det tilsvarende gen, i modsætning til en strengt celleautonom virkning, hvilket indebærer, at kun de celler, der udtrykker genet udviser specifik fænotype. Dette blev overbevisende demonstreret ved vævsspecifik ekspression eller vælte af de respektive proteiner i adskillige modeller for neurodegenerative sygdomme 18-26. Forskellige mekanismer er blevet foreslået som grundlag for denne ikke-celle autonome toksicitet i PMDs, herunder mindsket næringsstof forsyning, ubalance i neuronal signalering, glutamatexcitotoksicitet og neuroinflammation 16,27,28. Desuden et prion-lignende bevægelse af sygdomsspecifikke forbundet aggregater mellem celler might bidrage til dette aspekt 29,30. Stigende bevis antyder, at andre protein indeslutninger end prioner kan overføre fra celle til celle, hvilket kan forklare den karakteristiske spredning af patologi observeret i mange PMDs 30-36. Men det er endnu ikke afgjort, om der er en klar årsagssammenhæng mellem intercellulære bevægelse af sygdoms-proteiner og den toksiske effekt på de omkringliggende celler. Derfor er nødvendigt og væsentligt for udviklingen af ​​hidtil ukendte terapeutiske midler en bedre forståelse af de cellulære veje, der ligger til grund for celle-til-celle-transmission og ikke celleautonom toksicitet. Men mange aspekter af prion-lignende spredning og cellulære faktorer, der påvirker celle-til-celle-transmission af fejlfoldede proteiner i metazoans ikke godt forstået, navnlig i organismal niveau.

Nematoden Caenorhabditis elegans har flere fordele, der giver mulighed for at opdage nye facetter af prion-lignende spreading i metazoans 17. Det er transparent, hvilket muliggør in vivo sporing af fluorescens-mærkede proteiner i den levende organisme. Desuden er mange cellulære og fysiologiske processer er ramt af sygdom bevaret fra orme til menneske, og C. elegans er også modtagelig for en bred vifte af genetiske manipulationer og molekylære og biokemiske analyser 37-39. Nøjagtigt 959 somatiske celler udgør voksen hermafrodit med et simpelt organ plan, der stadig har flere forskellige vævstyper, herunder muskel, neuroner og tarmen.

At etablere en ny prion model C. elegans, valgte vi at eksogent udtrykker velkarakteriseret glutamin / asparagin (Q / N) -rige prion domæne nm af cytosoliske gær prionprotein Sup35, da der ikke er nogen kendte endogene prionproteiner i orme 4,40. Gær prioner har været uvurderlig i at belyse grundlæggende mekanismer i prion replikation 41-44. Endvidere NM er first cytosolisk prion-lignende protein, der har vist sig at rekapitulere det fulde livscyklus prion i pattedyrcellekultur 45,46. Ligeledes, når det udtrykkes i C. elegans, Sup35 prion domænet vedtaget bemærkelsesværdigt godt til de forskellige krav til opformering i metazoan celler sammenlignet med gærceller og udstillet centrale elementer i prion biologi 40. NM sammenlægning var forbundet med en dyb giftig fænotype, herunder afbrydelse af mitokondrie integritet og udseende forskellige autophagy relaterede blærer på celleniveau samt embryonale og larver anholdelse, forsinket udvikling, og en udbredt forstyrrelse af proteinfoldning miljøet på organismal niveau. Slående, prion domæne udviser celleautonom og ikke celleautonom toksicitet, der påvirker tilstødende væv, i hvilke transgenet ikke blev udtrykt. Endvidere er vesikulær transport af prion-domænet i og mellem celler overvåges tidstro <em> in vivo 40.

Her beskriver vi, hvordan at undersøge prion-lignende udbredelse i C. elegans. Vi vil forklare, hvordan at overvåge intra- og intercellulære transport af vesikler, der indeholder prion domæne ved hjælp tidsforskudt fluorescens mikroskopi. Vi vil lægge vægt på brugen af ​​vævsspecifikke folde sensorer og ubikvitært udtrykte stress journalister at evaluere celle autonome og ikke celle autonome virkninger på cellulær fitness. Endelig vil vi beskrive proceduren for en nylig udført genom bred RNA-interferens (RNAi) skærmen for at identificere nye modifikatorer af prion-induceret toksicitet. I kombination, kan disse metoder bidrage til drille hinanden genetiske veje er involveret i intercellulære bevægelse af proteiner og deres ikke celleautonom toksicitet.

Protocol

1. Overvågning transcellulær Spredning af Prion-lignende proteiner ved in vivo Time-lapse Imaging BEMÆRK: Grow C. elegans vildtype (WT) (N2) og transgene linjer ifølge standardmetoder og omhyggeligt kontrollere dyrkning temperatur 47. Generere transgene linjer C. elegans udtrykker prion-lignende protein, tagget med monomert rødt fluorescerende protein (mRFP). Se denne video, der viser, hvordan man bruger mikroinjektion 48. For …

Representative Results

Overvågning intercellulære spredning af prion-lignende proteiner ved in vivo time-lapse billeddannelse Transgene C. elegans linjer udtrykker prion domæne er særligt velegnede til analyse af visse aspekter af prion-lignende proteiner, fx celle-til-celle-transmission og ikke celleautonom toksicitet. Gennemsigtigheden af ​​dyrene muliggør sporing af fluorescens mærkede proteiner inde fra levende organisme i alle faser af livet. Ved at udnytt…

Discussion

De her beskrevne metoder hjælper til at illustrere spredning og komplekset celleautonom og ikke celleautonom toksicitet prion-lignende proteiner. Nylig opdagede vi, at en sammenlægning-tilbøjelige cytosoliske prion domæne optages i membranbundne vesikler i et autophagy relateret proces. En specifik undergruppe af disse vesikler transporterer prion domæne inden for og mellem celler og væv 40. Nøglen til at overvåge deres bevægelse i levende dyr er, at proteinet skal være mærket med mRFP, fordi kun m…

Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank Cindy Voisine and Yoko Shibata for helpful discussion and critical comments on the manuscript. We acknowledge the High Throughput Analysis Laboratory (HTAL) and the Biological Imaging Facility (BIF) at Northwestern University for their assistance. This work was funded by grants from the National Institutes of Health (NIGMS, NIA, NINDS), the Ellison Medical Foundation, and the Daniel F. and Ada L. Rice Foundation (to R.I.M.). C.I.N.-K. was supported by the Deutsche Forschungsgemeinschaft (KR 3726/1-1).

Materials

Reagent
Nanosphere size standards 100 nm ThermoScientific 3100A
Levamisole Sigma L-9756
IPTG Sigma 15502-10G
Ahringer RNAi library Source BioScience LifeSciences  http://www.lifesciences.sourcebioscience
.com/clone-products/non-mammalian/c-elegans/c-elegans-rnai-library/
Equipment
Sorvall Legend XTR Refrigerated Centrifuge, 120VAC ThermoScientific 75004521 http://www.coleparmer.com/Product/Thermo_Scientific_Sorvall_Legend_
XTR_Refrigerated_Centrifuge_120
VAC/EW-17707-60
96 pin replicator  Scionomix   http://www.scinomix.com/all-products/96-pin-replicator/
HiGro high-capacity, incubating shaker  Digilab http://www.digilabglobal.com/higro
Multidrop Combi Reagent Dispenser  Titertrek http://groups.molbiosci.northwestern.edu/hta/titertek.htm
Biomek FX AP96 Automated Workstation  Beckman Coulter http://groups.molbiosci.northwestern.edu/hta/biomek_multi.htm
Innova44 shaker New Brunswick http://www.eppendorf.com/int///index.php?sitemap=2.3&pb=d78efbc05310ec
04&action=products&contentid=1&
catalognode=83389
M205 FA  Leica http://www.leica-microsystems.com/de/produkte/stereomikroskope-makroskope/fluoreszenz/details/product/leica-m205-fa/
ORCA-R2 C10600-10BDigital CCD camera Hamamatsu http://www.hamamatsu.com/jp/en/community/life_science_camera/product/search/C10600-10B/index.html
Spinning Disc AF Confocal Microscope  Leica http://www.leica-microsystems.com/products/light-microscopes/life-science-research/fluorescence-microscopes/details/product/leica-sd-af/
Falcon 4M60 camera  Teledyne Dalsa  http://www.teledynedalsa.com/imaging/products/cameras/area-scan/falcon/PT-41-04M60/
Software
MetaMorph Microscopy Automation & Image Analysis Software Molecular Devices http://www.moleculardevices.com/products/software/meta-imaging-series/metamorph.html
Hamamatsu SimplePCI Image Analysis Software Meyer Instruments http://meyerinst.com/imaging-software/hamamatsu/index.htm
ImageJ NIH http://rsbweb.nih.gov/ij/download.html
wrMTrck plugin for ImageJ http://www.phage.dk/plugins/wrmtrck.html
C. elegans strains
N2 (WT) Caenorhabditis Genetics Center (CGC) http://www.cgc.cbs.umn.edu/strain.php?id=10570
AM815                                                    rmIs323[myo-3p::sup35(r2e2)::rfp] Morimoto lab available from our laboratory 
See table 1 for a source for folding sensor and stress reporter strains
DOWNLOAD MATERIALS LIST

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Tags

PrionPrion-like ProteinProtein AggregationProteostasisCaenorhabditis ElegansPrion DomainSup35Fluorescent TagGenetic ApproachesPrion PropagationCell-to-cell SpreadingProtein Misfolding DiseasesAlzheimer’s DiseaseParkinson’s DiseaseALSTransmissible Spongiform Encephalopathies
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Nussbaum-Krammer, C. I., Neto, M. F., Brielmann, R. M., Pedersen, J. S., Morimoto, R. I. Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans. J. Vis. Exp. (95), e52321, doi:10.3791/52321 (2015).
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