Name: Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans (Video) | JoVE Journal
Uploaded: 2015-01-08T14:00:00.000+00:00
Duration: 12 min 57 s
Description: Learn to examine prion-like protein spreading and toxicity using C. elegans as a model organism. This method employs time-lapse fluorescence microscopy and genome-wide RNAi screening to identify new toxicity modifiers.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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 mMNa2HPO4·7H2O, 22 mMKH2PO4, 86 mMNaCl, 1 mMMgSO4·7H2O, add dH2O up to 1 L).</li>
			<li>Allow them to hatch in M9 buffer with gentle agitation O/N at 20 °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 µl 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 µl nanosphere size standards solution (polystyrene beads, 100 nm) plus 3 µl 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 &amp; 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 &gt;600 nm.</li>
		<li>Open “MultiDimensional Acquisition”. Under the “Main” tab, check the boxes for “Timelapse” and “Run Journals” (Hardware Auto Focus: off).</li>
		<li>Under the “Saving” tab select or create the directory folder where the files should be saved. Assign a name to the file.</li>
		<li>Under the “Wave Length” tab select the appropriate illumination and adjust exposure and gain. Use “YokoQuad Red” (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 “Timelapse” tab select “Number of time points” = 301, “Duration” = 5 min and “Time/Interval” = 1 sec.</li>
		<li>Under the “Journals” tab select Journal: “AFC SET Z HOLD” and “AFC Return to Z HOLD”, Type: “Special” (twice), Initial Point: “Start of time point” and “End of time point”. This autofocus option is important to image the same section over a longer period of time.</li>
		<li>When the setup is done, press “Acquire”. The timelapse video is safed as separate TIFF files.</li>
		<li>Open all .tiff files of a given time series in ImageJ. Go to “Image” →“Stacks”→“Images to Stacks”. Optionally, adjust brightness and contrast, add size bar, etc. Export the movie under “File”→“Save as”→“AVI…” (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="RNAi experiment process, incubation steps, L1 worms, method: gene screening, diagram." src="/files/ftp_upload/52321/52321fig4highres.jpg" data-altupdatedat="1787630408000" data-alt="Figure 4"> 
Figure 4. 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 µl LB-amp media (50 μg/ml Ampicillin in LB) supplemented with 10% glycerol and inoculate using a 96-pin replicator. Grow at 37 °C O/N with agitation and store at -80 °C.</li>
		<li>Maintain C. elegans WT and transgenic lines at 20 °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 μg/ml ampicillin. Use an automated reagent dispenser to dispense (or pipette manually) 65 μl of the LB-amp media into each well of a 96 well plate.</li>
		<li>Remove 96 well Ahringer RNAi library plates from -80 °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 °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 °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 μl 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-β-D-thiogalactopyranosid (IPTG) to a 5 mM concentration in ddH2O. Use an automated workstation with a multichannel head to dispense (or pipet manually) 10 μl 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 °C.</li>
		<li>While bacteria are shaking, prepare the worms. Mix the M9/worm suspension well, and pipette a small sample (~5 - 10 µl) to a glass microscope slide. Count the number of worms under a stereomicroscope and calculate the number of worms per μl.</li>
		<li>Using sterile technique, prepare a solution of supplemented M9 (M9+) with the following final concentrations: 0.20 mg/ml IPTG, 8.0 μg/ml Cholesterol, 50 μg/ml Ampicillin, 9.6 μg/ml Tetracycline, 0.0835 μg/ml Fungizone, and 15 worms per 50 μl.
		<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 μl 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 °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 µg/ml ampicillin, 12.5 µg/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 µg/ml ampicillin, O/N at 37 °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 µl 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 in M9 media.</li>
		<li>Take a sample of the worm suspension and determine the amount of nematodes per µl 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 °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 “Live” 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 “Auto Expose” 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 “Time Scan” and choose a folder and a file name. Set “Field Delay” to 20 msec and “Stop at Time” to 30 sec. Press “Live Review” 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 “Start”.</li>
		<li>After the movie finishes recording, right click on the image and choose “Export Montage Sequence” 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 “Plugins” tab, then “wrmtrck” and select “wrMTrck Batch”. 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 “OK” 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 “*_labels.zip” files created and run the resulting “*_labels.tif” 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

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...

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.

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

The overall goal of the following experiments is to examine the spreading and complex toxicity of PreOn like proteins using the Metazoan model organism C elegance. This is achieved by monitoring the intra and intracellular transport of vesicles containing the PreOn like protein, using time-lapse fluorescence microscopy. Next tissue specific folding sensors and ubiquitously expressed stress.Reporters are used to evaluate cell autonomous and non-cell autonomous effects on cellular fitness. Then a genome-wide RNAi screen is performed in order to identify new modifiers of PreOn induced toxicity. The screen is performed in liquid culture using thrashing as a readout for toxicity.Finally, worm tracker, A plugin for image J is used to quantify movement of C elegance in combination. These methods allow teasing apart of genetic pathways involved in the intercellular movement of PreOn like proteins and their non-cell autonomous toxicity. After preparing synchronized sea elegance control and PreOn domain transgenic lines and 10%agros pads, according to the accompanying text protocol, pipette three microliters of 100 nanograms of polystyrene beads and three microliters of four millimolar limassol into the agros pad.Using a platinum wire pick transfer worms at the desired age from a plate into the solution and use a cover slip to cover to image the worms at the subcellular level. Place a slide into the microscope slide holder of a confocal microscope and use a 63 x or 100 x 1.4 NA oil objective. Next, open METAMORPH and select multidimensional acquisition.And under the main tab, check the boxes for time lapse and run journals. Under the saving tab, select or create the directory folder for saving files and assign a name to the file Under the wavelength tab, use Yoko quad red or an equivalent illumination of MRFP imaging and adjust the exposure to between 100 and 300 milliseconds and the camera gain to between 100 and 300. Next, under the time lapse tab, set time interval to one second, duration to five minutes, and the number of time points to 301.Under the journals tab. For journal select A FC set, Z hold, and A FC return to Z hold for type select special twice and for initial point, select start of time point and end of time point. When the setup is done, press acquire Following this procedure, other methods like particle tracking applications can be used in order to measure the dynamic properties of dis cus.After synchronizing prion domain transgenic and while type nematodes and copying the inger RNAI library plates according to the text protocol, incubate duplicated RNAi plates at 37 degrees Celsius and 300 RPM to use HT one 15 E coli bacteria harboring the empty vector L 4 4 4 0 as a control. Grow it separately in a culture tube and then use a multi-channel pipette to transfer 65 microliters of the culture into one quarter of the wells of 2 96. Well plates with an automated workstation containing a multi-channel head dispensed.10 microliters of five, millimolar of diluted IPTG into each well of the RNAi bacteria and control plates. Reseal them before incubating them for three hours while the bacteria are incubating. Mix synchronized wild type control and transgenic L one larvae and M nine buffer well and pipette five to 10 microliters onto a glass microscope.Slide count the worms and calculate the number of worms per microliter. After the three hour bacterial incubation, remove the plates from the incubator and allow them to come to room temperature to avoid heat stressing the worms. Next, supplement the M nine buffer with IPTG cholesterol and antibiotics according to the text protocol.And add the appropriate volume of PreOn domain transgenic worm solution to a final concentration of 15 worms per microliter mix to evenly distribute. Then dispense 50 microliters into each well of the RNAi plates and the wells of one of the empty vector control plates. Dispense wild type worm solution into the second control plate.Then leave all the plates unsealed to allow aeration and keep the liquid culture from evaporating by stacking four to five plates together, wrapping them with a damp paper towel followed by aluminum foil. Incubate the plates at 20 degrees Celsius and 200 RPM for four days. To the plates, use a Falcon four M 60 camera connected to a computer with a monitor to visually screen for increased thrashing as compared with control treated PreOn domain transgenic animals.The main advantage of using an automated liquid handling workstation to dispense the worms into the R RNAi plates is that it greatly reduces the variability and the number of worms per well. To perform a motility assay on solid plates and validate candidate RNAi clones grow RNAi bacteria overnight according to the text protocol. Then use one millimolar IPTG to induce the expression of DS RNA for three hours.Use 150 microliters of each RNAi bacterial clone to seed previously prepared NGM plates spreading into a thin layer. Allow the bacteria to dry for two days at room temperature in the dark under a stereo microscope. Determine the amount of synchronized worms and suspension as described earlier and add 25 to 30 L one larvae into each experimental plate.Incubate the nematodes at 20 degrees Celsius for four days until the worms reach. Day two of adulthood to quantitate worm motility, turn on the camera and the simple PCI imaging software. Click on the camera icon to enable adjustment of imaging conditions.Next, set up the imaging condition as follows. Set the gain to zero light mode to high speed index to one and binning to two. Then click live and auto expose and adjust the lighting conditions by moving the microscope mirrors and the brightness and contrast dials.Click on time, scan and type in a folder and a file name set field delay to 20 milliseconds and stop at time to 30 seconds. Press live review in order to choose the area of the plate to record. Then tap the plate three or four times on the stage.Quickly confirm its position in the field of view and press start after the movie finishes recording right click on the image and choose export montage sequence to export the movie from a CXD format to an A VI file. To analyze the motility videos, open image J choose the plugins tab, then choose worm tracker and select Worm Tracker batch, and select the directory containing all the files to be analyzed in the main input window of Worm Tracker batch load the input values as detailed here. An explanation for each of the parameters can be found in the instructions that accompany the plugin.Click okay and allow the movement analysis to run. Finally, to curate the results, confirm that all detected tracks are from actual sea elegance and eliminate artifacts. Open the TXT files created for each movie and copy the information to a data analysis software file.Open the labels zip files created and run the resulting labels TIFF to manually check and eliminate false worm tracks. Yesper Peterson, a former postdoc in the lab, first had the idea for the worm tracker as a substitute for our manual assessment of CL Elegance motil, which is a readout for toxicity. The worm tracker allows us to do these experiments in an automated way with no user bias while assessing more conditions in a shorter amount of time Using time-lapse imaging.The RFP tagged R two E two, which accumulates in tubular vesicles was seen transported within and between cells by coex expressing a folding sensor or stress reporter. Together with the PreOn like protein cell, autonomous and non-cell autonomous effects can be assessed. The expression of R two E two in muscle cells leads to an earlier onset of Q 44 aggregation in the intestine revealing that R two E two has non-cell autonomous effects on proteostasis co-expression of R two E two With the SOD three PGFP Transcriptional oxidative stress reporter leads to upregulation of the reporter in different tissues, demonstrating that the PreOn domain induces an oxidative stress response non-cell autonomously following the whole genome screen.This movie demonstrates that high resolution imaging and visualization on the computer screen enable the screening of one quarter of a plate simultaneously Once mastered, those screening protocol can be done in 12 rounds of 25 plates per session. If two sessions are performed per week, the entire RNAi library can be screened within six weeks. The worm tracker was used to quantify C elegance motility as seen here.Worm Tracker converts an original video into a binary format and each worm is assigned a track. Processed videos can be viewed with tracks superimposed to manually eliminate any artifacts. The body lengths per second parameter normalizes for potential size differences.Nematodes.This method identified 35 suppressors of PreOn induced toxicity that increased the motility phenotype to at least 133%and up to 256%compared to control treated worms. While leasing the worm tracker, it's important to remember to manually verify the obtain worm tracks. The software does a great job in identifying the worms, but sometimes the background can still be mistakenly identified as an animal.Therefore, remember to check your videos after running worm tracker on them. These methods can help answer key questions in the field of neurodegenerative diseases such as whether a certain disease link protein is able to spread from cell to cell, similar prence, or whether there is a link between the intercellular transmission and the complex toxicity of these proteins.Kids.

investigating-spreading-toxicity-prion-like-proteins-using-metazoan

Research

JoVE Journal

Biology

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

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

This video demonstrates the procedure for in vivo imaging of C. elegans expressing prion-like proteins tagged with red fluorescent protein. The protocol involves immobilizing nematodes on an agarose pad for time-lapse confocal microscopy, revealing protein aggregation and transport within and between cells. This technique is crucial for studying protein aggregation related to neurodegenerative diseases.

Time-Lapse Imaging to Monitor the Transcellular Spreading of Prion-Like Proteins in Transgenic Nematodes

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human Aβ42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

A Caenorhabditis elegans Model System for Amylopathy Study

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present a protocol for simple isolation of specific groups of live neuronal cells expressing green fluorescent protein from transgenic Caenorhabditis elegans lines. This method enables a variety of ex vivo studies focused on specific neurons and has the capacity to isolate cells for further short-term culturing.

Isolation of Specific Neuron Populations from Roundworm 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.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

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.

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

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.

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

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Generation of Stable Transgenic C. elegans Using Microinjection

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants.   This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Learn to examine prion-like protein spreading and toxicity using C. elegans as a model organism. This method employs time-lapse fluorescence microscopy and genome-wide RNAi screening to identify new toxicity modifiers.

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

Explore More Videos