Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

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

Many neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic lateral sclerosis (ALS), and transmissible spongiform encephalopathies (TSEs), are associated with aggregation-prone proteins and are hence collectively known as protein misfolding disorders (PMDs). TSEs or prion diseases constitute a unique class of PMDs in that they can be infectious in both humans and animals¹. At the molecular level, prions replicate by recruiting and converting monomeric α-helix-rich host-encoded cellular PrP (PrP^C) into the pathological β-sheet-rich PrP^Sc conformation^2,3. Sel....

Protocol

1. Monitoring Transcellular Spreading of Prion-like Proteins By In Vivo Time-lapse Imaging

NOTE: Grow C. elegans wild-type (WT) (N2) and transgenic lines according to standard methods and carefully control the cultivation temperature⁴⁷.

1. Generate transgenic lines of C. elegans expressing the prion-like protein, tagged with monomeric red fluorescent protein (mRFP). Watch this video that demonstrates how to use microinjection⁴⁸. For further details and methods describing how to integrate these extrachromosomal lines, see⁴⁹.
2. Prepare synchronized populations by egg....

Results

Monitoring intercellular spreading of prion-like proteins by in vivo time-lapse imaging

Transgenic C. elegans lines expressing the prion domain are particularly well suited for the analysis of certain aspects of prion-like proteins, e.g., cell-to-cell transmission and non cell autonomous toxicity. The transparency of the animals enables tracking of fluorescently tagged proteins from within the living organism at ever.......

Discussion

The methods described here help to illustrate spreading and the complex cell autonomous and non cell autonomous toxicity of prion-like proteins. We recently discovered that an aggregation-prone cytosolic prion domain is taken up into membrane-bound vesicles in an autophagy related process. A specific subset of these vesicles transports the prion domain within and between cells and tissues⁴⁰. The key to monitor their movement in the living animal is that the protein has to be tagged with mRFP, because only mRFP.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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