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).

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.
<ol>
	<li>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.</li>
	<li>Prepare synchronized populations by egg laying or bleaching according to standard methods50.
	<ol>
		<li>Synchronization by egg laying
		<ol>
			<li>Transfer 10 - 20 gravid adults on a plate and let them lay eggs for 1 - 2 hr. Remove adults from the plate and let the progeny grow until the desired age.</li>
		</ol>
		</li>
		<li>Synchronization by bleaching
		<ol>
			<li>Collect an unsynchronized population of gravid adults and bleach them with alkaline hypochlorite solution (250 mM NaOH and 1:4 (v/v) dilution of commercial bleach in H2O). Wash the eggs twice (218 x g for 1 min) with M9 buffer47 (21 mMNa2HPO4&#183;7H2O, 22 mMKH2PO4, 86 mMNaCl, 1 mMMgSO4&#183;7H2O, add dH2O up to 1 L).</li>
			<li>Allow them to hatch in M9 buffer with gentle agitation O/N at 20 &#176;C. Worm development will arrest at the L1 stage in the absence of a food source, leaving a synchronized population. Transfer L1s onto fresh Nematode Growth Media (NGM) plates seeded with OP50 E. coli bacteria and let the progeny develop until the desired age47.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Prepare 2% agarose pads (in H2O) on a microscope slide as described50.
	<ol>
		<li>Prepare two microscope slides with labeling tape placed over their entire length to be used as spacers. Place a third microscope slide between them.</li>
		<li>Dissolve 2% agarose in H2O and pipette one drop onto the clean slide.</li>
		<li>Place a fourth slide perpendicular to the three other slides on top of the agar drop. Gently press it down to flatten the pad to the same thickness as the labeling tape.</li>
		<li>Let it dry for 1 min before removing the spacers and gently pulling the slides apart. The agar pad will stick to one of them.</li>
	</ol>
	</li>
	<li>Pipette ~10 &#181;l anesthetic (2 mM levamisole in M9 buffer) to the pad and transfer ~10 animals using a platinum wire pick. Cover with a cover slip (~22 x 22 mm) and take images within 1 hr.</li>
	<li>Alternatively, to acquire movies over a longer period of time or to further reduce the possibility of any movement of the animals, use a combination of anesthetic and bead immobilization51.
	<ol>
		<li>Prepare 10% agarose pads (in M9 buffer) as described51 and add worms to 3 &#181;l nanosphere size standards solution (polystyrene beads, 100 nm) plus 3 &#181;l anesthetic (4 mM levamisole in M9 buffer). Cover gently with a cover slip. To avoid desiccation, seal the cover slip with VALAP (mixture of equal amounts of Vaseline, lanolin, and paraffin wax).</li>
	</ol>
	</li>
	<li>Image immobilized worms using a confocal microscope. 
	NOTE: Results are obtained using a Spinning Disc AF Confocal Microscope equipped with an EM-CCD camera and a Microscopy Automation &#38; Image Analysis Software such as MetaMorph and outline the specifics below, but other comparable confocal imaging systems can also be used.
	<ol>
		<li>Use the 63X or 100X/1.4NA oil objective and place the microscope slide containing worms into the microscope slide holder.</li>
		<li>Open the software. Adjust laser power and filters for mRFP imaging. Use the 561 nm laser at 10% power and emission filter &#62;600 nm.</li>
		<li>Open &#8220;MultiDimensional Acquisition&#8221;. Under the &#8220;Main&#8221; tab, check the boxes for &#8220;Timelapse&#8221; and &#8220;Run Journals&#8221; (Hardware Auto Focus: off).</li>
		<li>Under the &#8220;Saving&#8221; tab select or create the directory folder where the files should be saved. Assign a name to the file.</li>
		<li>Under the &#8220;Wave Length&#8221; tab select the appropriate illumination and adjust exposure and gain. Use &#8220;YokoQuad Red&#8221; (or an equivalent illumination setting for mRFP imaging) with an exposure between 100 and 300 msec and a camera gain between 100 and 300, depending on the individual sample (assessed by using the live image).</li>
		<li>Under the &#8220;Timelapse&#8221; tab select &#8220;Number of time points&#8221; = 301, &#8220;Duration&#8221; = 5 min and &#8220;Time/Interval&#8221; = 1 sec.</li>
		<li>Under the &#8220;Journals&#8221; tab select Journal: &#8220;AFC SET Z HOLD&#8221; and &#8220;AFC Return to Z HOLD&#8221;, Type: &#8220;Special&#8221; (twice), Initial Point: &#8220;Start of time point&#8221; and &#8220;End of time point&#8221;. This autofocus option is important to image the same section over a longer period of time.</li>
		<li>When the setup is done, press &#8220;Acquire&#8221;. The timelapse video is safed as separate TIFF files.</li>
		<li>Open all .tiff files of a given time series in ImageJ. Go to &#8220;Image&#8221; &#8594;&#8220;Stacks&#8221;&#8594;&#8220;Images to Stacks&#8221;. Optionally, adjust brightness and contrast, add size bar, etc. Export the movie under &#8220;File&#8221;&#8594;&#8220;Save as&#8221;&#8594;&#8220;AVI&#8230;&#8221; (for an example, see Video 1 and 2 corresponding to Figure 1).</li>
	</ol>
	</li>
</ol>
2. Using Folding Sensors and Stress Reporters to Investigate Cell Autonomous and Non cell Autonomous Affects on Proteostasis and Toxicity
<ol>
	<li>Generate transgenic lines that coexpress a folding sensor or stress reporter together with the prion-like protein. For methods on how to establish crosses or generate transgenic lines, see48,49,52. See Table 1 for a list of C. elegans strains that can be used.</li>
	<li>Prepare synchronized populations of transgenic animals and grow them until the desired age as described above (section 1.2)50.</li>
	<li>Using a stereomicroscope, examine the respective phenotype of the folding sensor or stress reporter.
	<ol>
		<li>For the folding sensor, determine the number of animals harboring aggregates on each day after synchronization (for an example, see Figure 2E and F).</li>
		<li>For the stress reporter, test if the coexpression of the prion-like protein results in an increased fluorescence of the reporter (for an example, see Figure 3).</li>
	</ol>
	</li>
</ol>
3. Genome-wide Screen for Suppression of Prion-induced Toxicity in C. elegans
<img alt="Figure 4" src="/files/ftp_upload/52321/52321fig4highres.jpg" /> 
Figure 4.&#160;Schematic representation of the RNAi screening protocol. See protocol section 3 for a detailed description of the individual steps.
<ol>
	<li>Synchronization of C. elegans worms and duplication of RNAi library
	<ol>
		<li>For the RNAi screen, use the Ahringer RNAi library (or the Vidal RNAi library)53,54. Rearray the RNAi library from the original 384 well format into 96 well plates by filling the plates with 100 &#181;l LB-amp media (50 &#956;g/ml Ampicillin in LB) supplemented with 10% glycerol and inoculate using a 96-pin replicator. Grow at 37 &#176;C O/N with agitation and store at -80 &#176;C.</li>
		<li>Maintain C. elegans WT and transgenic lines at 20 &#176;C on NGM plates seeded with OP50 E. coli bacteria according to standard methods47.</li>
		<li>Synchronize prion domain transgenic and WT (control) nematodes by bleaching (see section 1.2).</li>
		<li>On the same day that the worms are bleached, prepare LB media supplemented with 50 &#956;g/ml ampicillin. Use an automated reagent dispenser to dispense (or pipette manually) 65 &#956;l of the LB-amp media into each well of a 96 well plate.</li>
		<li>Remove 96 well Ahringer RNAi library plates from -80 &#176;C and bring them to a sterile hood lined with paper towels. Immediately remove the seal-tape by holding the plate upside down while plates are still frozen, being careful to avoid contamination from ice on the outside of the plates. Reinvert the plates and let them thaw for about 30 min.</li>
		<li>Dip one sterile 96 pin replicator into one RNAi library plate, and then into one fresh LB-amp plate. Use a fresh sterile replicator for each plate. Seal all plates with adhesive foil tape and return the library plates to -80 &#176;C. The replicators can be soaked in bleach, rinsed, and autoclaved to be used again.</li>
		<li>Allow duplicated RNAi plates to grow O/N in an incubator set to 37 &#176;C and 300 rpm. To use HT115 E. coli bacteria harboring the empty vector (L4440) as a control, grow it separately in a culture tube and then pipette 65 &#956;l of the culture with a multichannel pipette into one quarter of two 96 well plates.</li>
	</ol>
	</li>
	<li>Induction of bacterial dsRNA production and addition of worms
	<ol>
		<li>Dilute Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) to a 5 mM concentration in ddH2O. Use an automated workstation with a multichannel head to dispense (or pipet manually) 10 &#956;l of diluted IPTG into each well of the RNAi bacteria and the control plates. Reseal and place the plates back into the incubator. Let them shake for 3 hr at 37 &#176;C.</li>
		<li>While bacteria are shaking, prepare the worms. Mix the M9/worm suspension well, and pipette a small sample (~5 - 10 &#181;l) to a glass microscope slide. Count the number of worms under a stereomicroscope and calculate the number of worms per &#956;l.</li>
		<li>Using sterile technique, prepare a solution of supplemented M9 (M9+) with the following final concentrations: 0.20 mg/ml IPTG, 8.0 &#956;g/ml Cholesterol, 50 &#956;g/ml Ampicillin, 9.6 &#956;g/ml Tetracycline, 0.0835 &#956;g/ml Fungizone, and 15 worms per 50 &#956;l.
		<ol>
			<li>Make separate solutions for prion domain transgenic and WT worms. The final volume of M9+ worm solution needed will depend on the number of RNAi plates copied (see below), plus ~30 ml of dead volume that will remain in the reservoir, if an automated dispenser is used.</li>
		</ol>
		</li>
		<li>After the 3 hr induction of bacterial dsRNA production, take the plates out of the incubator and let them cool to RT (~30 min) to avoid heat stressing the animals.</li>
		<li>Dispense 50 &#956;l M9+ prion domain transgenic worm solution to each well of the RNAi plates and one of the empty vector control plates. Make sure to mix the worm solution before each step as the animals tend to sediment to the bottom of the reservoir.</li>
		<li>Dispense WT worms into the second control plate. Leave the plates unsealed to allow aeration. To keep the liquid culture from evaporating, stack 4 - 5 plates together and wrap with a damp paper towel and aluminum foil. Incubate at 200 rpm at 20 &#176;C for 4 days.</li>
	</ol>
	</li>
	<li>Scoring 
	NOTE: After 4 days in the incubator, the worms will be at the second day of adulthood and are ready to screen. Let the animals adjust to non shaking conditions for 30 min before screening to ensure undisturbed thrashing.
	<ol>
		<li>Using a Falcon 4M60 camera connected to a computer with a monitor, screen visually for increased thrashing as compared with control treated prion domain transgenic animals.</li>
		<li>Compile a list of preliminary hits to confirm using wrMTrck (see next section).</li>
	</ol>
	</li>
</ol>
4. Confirmation of Preliminary Screen Hits
<ol>
	<li>Motility assay on solid plates
	<ol>
		<li>Prepare plates with NGM supplemented with 100 &#181;g/ml ampicillin, 12.5 &#181;g/ml tetracycline and 1 mM IPTG, according to standard methods47. If possible, use a plate-pouring machine to assure all plates have the same height of media, which will allow for a more streamlined video acquisition process.</li>
		<li>Grow the different RNAi clones in ~1 ml LB + 50 &#181;g/ml ampicillin, O/N at 37 &#176;C and 250 rpm.</li>
		<li>The next day, induce the expression of the dsRNA with 1 mM IPTG for 3 hr. Seed the plates with 150 &#181;l of each RNAi bacterial clone, spread into a thin layer. Let the bacteria dry for 2 days at RT in the dark. Prepare 3 plates per RNAi clone.</li>
		<li>Synchronize the worm population by bleaching according to standard methods50 (see section 1.2), and let the eggs hatch O/N&#160;in M9 media.</li>
		<li>Take a sample of the worm suspension and determine the amount of nematodes per &#181;l at the stereomicroscope. Then, pipette the proper volume of M9 plus worms into each experimental plate so that it contains 25 - 30 L1 worms. Grow the nematodes at 20 &#176;C for 4 days until the worms reach day 2 of adulthood.</li>
	</ol>
	</li>
	<li>Quantitative analysis of worm motility with the wrMTrck plugin for ImageJ 
	NOTE: The videos were recorded using a stereomicroscope at 10X magnification with a Hamamatsu Orca-R2 digital camera C10600-10B and the Hamamatsu Simple PCI imaging software.
	<ol>
		<li>Turn on the camera and the Simple PCI imaging software. Click on &#8220;Live&#8221; to allow for adjustment of the imaging conditions.</li>
		<li>Set up the video conditions as follows: Gain = 0; Light Mode = High; Speed Index = 1; Binning = 2. Click &#8220;Auto Expose&#8221; and then adjust the lighting conditions, moving the microscope mirrors and the brightness and contrast dials. 
		NOTE: The video needs to have a high contrast without being overexposed, so that the animals appear as black shapes on a bright background.</li>
		<li>Click on &#8220;Time Scan&#8221; and choose a folder and a file name. Set &#8220;Field Delay&#8221; to 20 msec and &#8220;Stop at Time&#8221; to 30 sec. Press &#8220;Live Review&#8221; in order to choose the area of the plate to record (where most worms are). Tap the plate 3 or 4 times on the stage, quickly confirm its position in the field of view and press &#8220;Start&#8221;.</li>
		<li>After the movie finishes recording, right click on the image and choose &#8220;Export Montage Sequence&#8221; to export the movie from a .cxd format to an .avi.</li>
	</ol>
	</li>
	<li>Analyzing the motility videos
	<ol>
		<li>Open the ImageJ software, go to the &#8220;Plugins&#8221; tab, then &#8220;wrmtrck&#8221; and select &#8220;wrMTrck Batch&#8221;. Select the directory containing all the files to be analyzed.</li>
		<li>In the main input window of wrMTrck_Batch, load the input values as detailed in Figure 5C. Explanation for each of the parameters can be found in the instructions that accompany the plugin. Click &#8220;OK&#8221; and let the movement analysis run.</li>
		<li>To curate the results and confirm that all detected tracks are from actual C. elegans and eliminate artifacts, open each of the .txt files created for each movie and copy the information to a data analysis software file. Open the &#8220;*_labels.zip&#8221; files created and run the resulting &#8220;*_labels.tif&#8221; to manually check and eliminate false worm tracks.</li>
	</ol>
	</li>
</ol>

The authors declare no competing financial interests.

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...

Many neurodegenerative diseases, including Alzheimer&#8217;s disease (AD), Parkinson&#8217;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 &#945;-helix-rich host-encoded cellular PrP (PrPC) into the pathological &#946;-sheet-rich PrPSc conformation2,3. Self-propagating protein aggregates have been also identified in fungi, which share important characteristics with mammalian prions4,5. Additionally, mammalian prions are capable of moving from cell-to-cell and infect na&#239;ve cells6,7.
While PMDs other than TSEs are not infectious, they share a common pathogenic principle with prion diseases8,9. Although the proteins linked to each of the PMDs are not related in structure or function, they all form aggregates via a crystallization-like process called nucleated or seeded polymerization; moreover proteinaceous seeds grow by recruiting their soluble isoforms2,10,11. The efficiency to self-propagate varies in vivo, depending on the intrinsic properties of the protein, which together with additional cellular factors such as molecular chaperones ultimately determine rates of aggregate nucleation, seeding, fragmentation and spreading12-15. Hence, there must exist a fine balance among these factors that allows efficient propagation of protein aggregation. This might also explain why only some amyloidogenic aggregates harbor the characteristics of a prion, and thus not all PMDs are infectious. Prions seem to represent &#8216;top-performers&#8217; of a wide spectrum of self-replicating proteinaceous aggregates, which makes them a powerful tool to study PMDs8,13.
Intriguingly, the toxicity associated with disease-related aggregates often has a non cell autonomous component16,17. This means that they affect neighboring cells that do not express the corresponding gene, in contrast to a strictly cell autonomous effect, which implies that only the cells expressing the gene exhibit the specific phenotype. This was compellingly demonstrated by tissue-specific expression or knock down of the respective proteins in numerous models of neurodegenerative diseases18-26. Various mechanisms have been suggested as a basis for this non-cell autonomous toxicity in PMDs, including diminished nutrient supply, imbalance in neuronal signaling, glutamate excitotoxicity, and neuroinflammation16,27,28. In addition, a prion-like movement of disease-linked aggregates between cells might contribute to this aspect29,30. Increasing evidence suggests that protein inclusions other than prions can transmit from cell-to-cell, which may explain the characteristic spreading of pathology observed in many PMDs30-36. However, it has yet to be determined whether there is a clear causal link between intercellular movement of disease proteins and the toxic effect on neighboring cells. Therefore, a better understanding of the cellular pathways that underlie cell-to-cell transmission and non cell autonomous toxicity is necessary and essential for the development of novel therapeutics. However, many aspects of prion-like spreading and cellular factors that influence cell-to-cell transmission of misfolded proteins in metazoans are not well understood, in particular at the organismal level.
The nematode Caenorhabditis elegans has several advantages that provide the potential to discover new facets of prion-like spreading in metazoans17. It is transparent, allowing for in vivo tracking of fluorescently tagged proteins in the living organism. Furthermore, many cellular and physiological processes affected by disease are conserved from worms to human, and C. elegans is also amenable to a wide variety of genetic manipulations and molecular and biochemical analyses37-39. Exactly 959 somatic cells make up the adult hermaphrodite with a simple body plan that still has several distinct tissue types, including muscle, neurons and intestine.
To establish a new prion model in C. elegans, we chose to exogenously express the well characterized glutamine/asparagine (Q/N)-rich prion domain NM of the cytosolic yeast prion protein Sup35, since there are no known endogenous prion proteins in worms4,40. Yeast prions have been invaluable in elucidating basic mechanisms of prion replication41-44. Furthermore, NM is the first cytosolic prion-like protein that has been shown to recapitulate the full life cycle of a prion in mammalian cell culture45,46. Likewise, when expressed in C. elegans, the Sup35 prion domain adopted remarkably well to the different requirements for propagation in metazoan cells compared to yeast cells and exhibited key features of prion biology40. NM aggregation was associated with a profound toxic phenotype, including the disruption of mitochondrial integrity and appearance of various autophagy related vesicles on the cellular level, as well as embryonic and larval arrest, developmental delay, and a widespread disturbance of the protein folding environment on the organismal level. Strikingly, the prion domain exhibits cell autonomous and non cell autonomous toxicity, affecting neighboring tissues in which the transgene was not expressed. Furthermore, the vesicular transport of the prion domain within and between cells is monitored real time in vivo40.
Here we describe how to examine prion-like dissemination in C. elegans. We will explain how to monitor the intra- and intercellular transport of vesicles containing the prion domain using time-lapse fluorescence microscopy. We will emphasize the use of tissue-specific folding sensors and ubiquitously expressed stress reporters to evaluate cell autonomous and non cell autonomous effects on cellular fitness. Finally, we will describe the procedure of a recently performed genome wide RNA interference (RNAi) screen to identify new modifiers of prion-induced toxicity. In combination, these methods can help to tease apart genetic pathways involved in the intercellular movement of proteins and their non cell autonomous toxicity.

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

investigating the spreading and toxicity of prion-like proteins using the metazoan model organism c. elegans

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&#8217;s disease (AD), Parkinson&#8217;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.

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...

Watch this Scientific Journal Video about Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans at JoVE.com

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

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.

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

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

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15.8K Views.  Northwestern University. 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.

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

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

Neuroscience

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis ...

Biochemistry

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

Immunology and Infection

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. Here, a Drosophila melanogaster model that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach we describe here can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent&#160;model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a&#160;knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::G&#945;S(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

In a C. elegans excitotoxicity model, this protocol employs in vivo imaging to analyze the regulation of necrotic neurodegeneration, the effect of genes encoding candidate mediators, and involvement of mitochondria. Cell dissociation and sorting is used to specifically obtain at-risk neurons for cell-specific transcriptomic analysis of neurodegeneration and neuroprotection mechanisms.

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the ...

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s disease (HD) is principally driven by a CAG nucleotide repeat that encodes an expanded glutamine tract in huntingtin protein. Thus, the inhibition of polyglutamine (polyQ) aggregation and, in particular, aggregation-associated neurotoxicity is a useful strategy for the prevention of HD and other polyQ-associated conditions. This paper introduces generalized experimental protocols to assess the neuroprotective capacity of test compounds against HD using established polyQ transgenic Caenorhabditis elegans models. The AM141 strain is chosen for the polyQ aggregation assay as an age-associated phenotype of discrete fluorescent aggregates can be easily observed in its body wall at the adult stage due to muscle-specific expression of polyQ::YFP fusion proteins. In contrast, the HA759 model with strong expression of polyQ-expanded tracts in ASH neurons is used to examine neuronal death and chemoavoidance behavior. To comprehensively evaluate the neuroprotective capacity of target compounds, the above test results are ultimately presented as a radar chart with profiling of multiple phenotypes in a manner of direct comparison and direct viewing.

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Here, we present a protocol to assess the neuroprotective activities of test compounds in Caenorhabditis elegans, including polyglutamine aggregation, neuronal death, and chemoavoidance behavior, as well as an exemplary integration of multiple phenotypes.

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...

Tissue-Specific RNAi Tools to Identify Components for Systemic Stress Signaling

Over the past decade there has been a transformative increase in knowledge surrounding the regulation of protein quality control processes, unveiling the importance of intercellular signaling processes in the regulation of cell-nonautonomous proteostasis. Recent studies are now beginning to uncover signaling components and pathways that coordinate protein quality control from one tissue to another. It is therefore important to identify mechanisms and components of the cell-nonautonomous proteostasis network (PN) and its relevance for aging, stress responses and protein misfolding diseases. In the laboratory, we use genetic knockdown by tissue-specific RNAi in combination with stress reporters and tissue-specific proteostasis sensors to study this. We describe methodologies to examine and to identify components of the cell-nonautonomous PN that can act in tissues perceiving a stress condition and in responding cells to activate a protective response. We first describe how to generate hairpin RNAi constructs for constitutive genetic knockdown in specific tissues and how to perform tissue-specific genetic knockdown by feeding RNAi at different life stages. Stress reporters&#160;and behavioral assays function as&#160;valuable readouts&#160;that enable&#160;the fast screening of genes and conditions modifying systemic stress signaling processes. Finally, proteostasis sensors expressed in different tissues are utilized to determine changes in the tissue-specific capacity of the PN at different stages of development and aging. Thus, these tools should help clarify and allow monitoring the capacity of PN in specific tissues, while helping to identify components that function in different tissues to mediate cell-nonautonomous PN in an organism.

Maintenance of organismal proteostasis requires the coordination of protein quality control responses such as chaperone expression from one tissue to another. Here, we provide tools used in C. elegans that allow monitoring of proteostasis capacity in specific tissues and determine intercellular signaling responses.

Over the past decade there has been a transformative increase in knowledge surrounding the regulation of protein quality control processes, unveiling the ...

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

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