We would like to thank past and present members of the Samuelson laboratory for their assistance in the refinement of this method and/or discussion that aided the development of this manuscript. Research reported in this publication was supported by the National Institute on Aging of the National Institutes of Health under Award Numbers RF1AG062593 and R21AG064519. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

1. Preparation of reagents
<ol>
	<li>Select genes of interest to be inactivated via feeding-based RNAi. Purchase stocks of HT115 E. coli containing the RNAi clone of interest20. Alternatively, subclone the cDNA of the gene of interest into the multicloning site of the L4440 plasmid. 
	NOTE: To prevent degradation of dsRNA within the bacteria, use the HT115 strain. This is an RNase III-deficient E. coli strain with IPTG-inducible T7 polymerase activity. For proteostasis studies that do not use feeding-based RNAi, either HT115 or OP50 E. coli on standard NGM plates can be used.</li>
	<li>Prepare 5 x 6 cm plates per test condition. For experiments using RNAi, induce dsRNA production in transformed HT115 E. coli. For studies without RNAi, OP50 E. coli on standard NGM plates can be used (See Supplemental File 1 for standard NGM and RNAi plate recipes). 
	NOTE: RNAi plates can be stored at 4 &#176;C for several months before seeding with bacteria.</li>
	<li>Grow cultures of E. coli overnight (16-20 h) at 37 &#176;C in a shaking incubator at 220 rpm. Grow HT115 E. coli in Luria Broth (LB) with ampicillin (50 &#956;g/mL). 
	NOTE: Standard OP50 E. coli is not antibiotic resistant, but streptomycin resistant variants are also available.
	<ol>
		<li>Concentrate bacteria by centrifugation at 2,400 x g for 15-20 min in a benchtop centrifuge, aspirate the supernatant, and re-suspend the pellet in 1/10th the starting volume (i.e., 10x concentration) of LB with ampicillin for HT115, or LB without ampicillin for standard OP50.</li>
		<li>Aliquot 200 &#181;L of concentrated 10x bacteria to each plate (3-4 replicates per test condition, with 2 extra backup plates, to be used in the event of contamination).</li>
		<li>Allow open plates to dry in a clean environment such as a laminar flow bench until all liquid has been absorbed or allow covered plates to dry on a lab bench overnight.</li>
	</ol>
	</li>
	<li>For seeded RNAi plates dried in a hood, store dried plates within a worm box overnight (up to 24 hours) at room temperature. After 1 day at RT, plates can be stored at 4 &#176;C for up to 2 weeks in a sealed bag (to prevent plates from drying out). Before use, allow plates stored at 4 &#176;C to return to room temperature within the zip-lock bag to prevent condensation from introducing airborne fungal contaminates.
	<ol>
		<li>When using feeding-based RNAi, leave seeded HT115 bacteria on RNAi plates at room temperature for at least 12 hours prior to adding C. elegans. RNAi plates contain IPTG, which induces dsRNA production. Alternatively, IPTG can be added to the liquid cultures at the end of step 1.3.1, 2 hours prior to seeding.</li>
		<li>Avoid long-term storage of seeded HT115 plates at room temperature (greater than a few days) to avoid cracking of the agar that will cause C. elegans to burrow into agar.</li>
	</ol>
	</li>
</ol>
2. Synchronization of C. elegans
NOTE:&#160;Choose whether to synchronize C. elegans by either alkaline hypochlorite treatment of gravid adults or egg lay.
<ol>
	<li>Synchronize animals by hypochlorite treatment of gravid adult animals to promote the release of embryos22.
	<ol>
		<li>For hypochlorite treatment, wash gravid hermaphrodites 2 times with M9 buffer, then resuspend in 5 mL hypochlorite solution (3.25 mL of hypochlorite solution, 1 mL of M9 buffer and 0.75 mL of 5 M NaOH) for 5 min, shaking resuspended animals every minute. After 5 min, spin down animals and wash 3 times with M9 buffer. 
		NOTE: Hypochlorite treatment has been posited to affect proteostasis21.</li>
		<li>After hypochlorite treatment, allow embryos to hatch overnight in 3 mL of M9 solution with rotation at 20 &#176;C.</li>
		<li>Calculate the density of L1 animals (or alternatively, embryos) per &#181;L by dropping 10 &#181;L of L1 solution 3x onto a 6 cm plate and counting the number of L1 animals to calculate the average number of L1 animals per &#181;L. L1 animals will settle over time. Therefore, periodically mix L1 solutions to avoid settling.</li>
		<li>Seed 50 L1 animals per plate, count and record the number seeded, and move plates to a 20 &#176;C incubator. It is important to know the actual number of animals on each plate at the start of the assay. 
		NOTE: Synchronizing L1 animals by overnight hatching in M9 is routinely used as it minimizes developmental heterogeneity after placing animals onto food. However, synchronization occurs through developmental arrest in response to starvation cues, which for some studies should be avoided (see23 for additional discussion). As an alternative, roughly synchronized animals can be obtained through an egg-lay, see 2.2.</li>
		<li>Freeze and store leftover L1 animals in liquid nitrogen or a -80 &#176;C freezer. In this way, a sample of each strain at the time of the experimental setup is preserved, creating a valuable resource for future studies and improving reproducibility. 
		NOTE: See&#160;Supplemental File 1&#160;for hypochlorite and M9 solution recipes.</li>
	</ol>
	</li>
	<li>To synchronize animals via egg lay, place 20&#160;young gravid adult animals (day 1 of adulthood) onto each plate for 4-6 hours, and allow them to lay eggs until there are approximately 50 eggs per plate.&#160;Remove all gravid adults and move plates with eggs to a 20 &#176;C incubator. 
	NOTE: The gravid adult animals should be synchronized at the first day of adulthood. Older animals may lay eggs that have been retained in the uterus, causing the release of eggs that are in a more advanced developmental stage.</li>
</ol>
3. Progeny production
NOTE: Steps must be taken to either prevent progeny production or to separate the synchronized starting population from their progeny. Preventing progeny production can be achieved chemically with addition of 5-Fluoro-2&#39;-deoxyuridine (FUdR) to plates, which is described here. Some studies have reported that FUdR can alter proteostasis24,25. Alternative approaches to prevent progeny production are discussed below.
<ol>
	<li>Make a 1000x stock solution of FUdR by dissolving 1 g of FUdR into 10 mL of ultrapure H2O. Filter sterilize stock FUdR with a 0.2 &#956;m filter and a 10 mL syringe. Aliquot 1 mL of stock into a sterile 1.5 mL tube. Freeze and store at -20 &#176;C.</li>
	<li>Grow animals at 20 &#176;C until the L4 stage. N2 animals raised from synchronized L1s from hypochlorite treatment require approximately 40 hours of growth at 20 &#176;C to reach L4. Growth rates for other strains should be empirically tested prior to experimentation. For details on how to accurately stage C. elegans see26.
	<ol>
		<li>To each 6 cm plate with L4 animals, add 100 &#181;L of 80x FUdR. It is critical to add FUdR at the L4 stage.</li>
	</ol>
	</li>
	<li>Return plates to a worm box and put the box in a zip-lock bag. Return to appropriate incubator.</li>
</ol>
4. Measuring the decline in proteostasis in muscle tissue by using polyglutamine-expressing animals
NOTE: Two methods can be used to identify proteostasis decline in muscle cells: imaging the formation of protein aggregates during aging (4.1) and measuring the proteotoxicity these aggregates cause with age through the onset of paralysis (4.2).
<ol>
	<li>Imaging polyglutamine aggregate formation in the muscle during aging 
	NOTE: The age-dependent progression of protein aggregation in the muscle cells is imaged using polyglutamine (polyQ) repeats fused to a yellow fluorescent protein (YFP). This protocol outlines use of strain AM140 rmIs132[unc-54p::Q35::YFP], but other polyglutamine variant strains can also be used. The polyQ::YFP transgene is expressed in the muscle using the unc-54 muscle-specific promoter. Synchronized unc-54p::polyQ::YFP expressing animals are visualized at Day 1, 2, 3 and 4 of adulthood. To visualize unc-54p::polyQ::YFP aggregates, use a compound microscope equipped for fluorescence or a fluorescent dissecting microscope. AM140 is available from the Caenorhabditis Genetics Center (CGC) at: https://cgc.umn.edu/strain/AM140.
	<ol>
		<li>On imaging days (Day 1, 2, 3 and 4 of adulthood), pick 20 animals and mount on a microscope slide setup with a 3% agarose pad and a 5 &#956;L drop of 10 mM sodium azide (diluted in M9 buffer).</li>
		<li>After all animals&#160;are immobilized by sodium azide (~ 5 minutes), image the whole bodies of the animals using a 10x magnification lens (Figure 1A). For imaging, use a FITC filter and the same exposure for every animal. Discard slides after imaging. 
		NOTE: Alternatively, YFP foci can also be quantified directly on plates, but movement must be inhibited by applying a stream of carbon dioxide onto the plate while scoring. This alternative method is most appropriate when screening a large number of conditions (see discussion for more details).</li>
		<li>After acquisition of images, count the number of foci in the body wall muscles of the whole animal. Foci are brighter punctuated signals that can be differentiated from the dimmer soluble signal in the background.</li>
		<li>Plot the progression of YFP foci accumulation from days 1, 2, 3 and 4. Plot an XY graph where X represents days of adulthood and Y represents number of unc-54p::polyQ::YFP foci (Figure 1B). The number of foci in the experimental condition (e.g., gene downregulation or overexpression) is compared to control animals. Perform statistical analysis between the groups at each time point observed for each trial.
		<ol>
			<li>When comparing foci counts for only two conditions, perform an independent sample t-test at each time point.</li>
			<li>When comparing foci counts for more than two conditions, perform an omnibus one-way ANOVA analysis for each time point, including the data for all the conditions at that time point. If the omnibus ANOVA yields a significant p-value (i.e., p &#60; 0.005), perform a post-hoc analysis with pairwise independent sample t-tests to determine the specific conditions between which a significant difference in foci was detected (e.g., for groups A, B, and C compare A &#38; B, A &#38; C, and B &#38; C).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Measuring animal paralysis rates as a surrogate for polyglutamine toxicity in muscle cells 
	NOTE: The age-dependent increase of polyQ aggregates in muscle causes the decline of muscular function that drives locomotion. This defect in locomotion can be determined by measuring the progressive rate of paralysis in polyQ-expressing animals. For the paralysis assay, synchronized unc-54p::polyQ::YFP expressing animals are scored at Day 3, 5, 7 and 9 of adulthood. Add additional time points, as needed to extend the scoring range, such that you can assess the effect of genetic perturbation.
	<ol>
		<li>On scoring days (Day 3, 5, 7 and 9 of adulthood), look at the 6 cm plates containing 50 animals and record the number of paralyzed animals. Remove paralyzed animals from the plate. If animals have crawled off the plate, or have died, they should be censored; record the event so it can be accounted for in the analysis. 
		NOTE: An animal is considered to be paralyzed when no movement is observed after exposing them to light or gentle touch stimuli. Pharyngeal pumping activity is used to determine whether nonmoving animals are alive or dead.</li>
		<li>At the completion of the experiment, calculate the paralysis rate for each condition. Do this at each time point by dividing the fraction of paralyzed animals by the total number of animals observed for that condition at that time. If observing multiple plates per condition per time point (i.e., replicate plates), calculate the ratio separately for each replicate.</li>
		<li>Plot the progression of paralysis rate in an XY graph (scatterplot). X represents days of adulthood and Y represents paralysis rates. The paralysis rate of unc-54p::polyQ::YFP animals is compared to control animals (Figure 1C). Perform statistical analysis between the groups; for multiple trials, treat trials independently. Use the Cox Proportional-Hazards Regression and the Wald test. Most data analysis tools that support survival analysis also support Cox modeling.
		<ol>
			<li>Transform the data: each condition should have two columns, one for time, and another for &#8220;event&#8221;. At each time point observed, add a row with &#8220;0&#8221; for each paralyzed animal, and a &#8220;1&#8221; for each censored animal. The total number of rows should be equal to the number of starting animals on the plate for that condition.</li>
			<li>Perform Cox Proportional-Hazards modeling, followed by the Wald test, according to instructions for your statistical software. A univariate model will be appropriate for typical experiment designs. For more than two conditions, if a test with all conditions included indicates a significant result (i.e., p &#60; 0.005), pairwise tests between conditions can be performed to determine the specific significant pair(s) of conditions. 
			NOTE: The Cox model relies on an assumption that the test condition(s) modulate the probability of an event proportionally over time. This means, for example, that the Cox model would not be appropriate for comparing a condition under which most animals are paralyzed at day 1 to a condition under which most animals are paralyzed at day 9. Extensions of the Cox model, and other approaches for comparing proportions paralyzed at each time point, are available for cases where the proportional hazards assumption does not hold, see27,28,29,30.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Measuring the decline in proteostasis in neuronal tissue by using polyglutamine expressing animals.
NOTE: Two complementary methods are used to assay proteostasis decline in neurons (1) by quantifying the formation of protein aggregates (fluorescent foci) during aging and (2) by measuring age-associated decline in the neuronal proteome via a movement-based assay.
<ol>
	<li>Imaging polyglutamine aggregate formation in the neurons during aging 
	NOTE: Similar to the assay already discussed in muscle tissue, the age-dependent progression of protein aggregation in the neurons is imaged by expressing a polyQ::YFP fusion protein driven by the pan-neuronal tissue-specific promoter of rgef-1. Specifically we use AM101: rmIs110 [rgef-1p::Q40::YFP], a forty glutamine fusion to YFP16. To visualize rgef-1p::polyQ::YFP, use a compound microscope equipped for fluorescence with a 40x magnification lens. AM101 is available from the CGC at: https://cgc.umn.edu/strain/AM101.
	<ol>
		<li>On imaging days (Day 4, 6, 8 and 10 of adulthood), pick 20 animals and mount on a microscope slide containing a 3% agarose pad with a 5 &#956;L drop of 10 mM sodium azide (diluted in M9 buffer).</li>
		<li>After all animals are immobilized by sodium azide (~ 5 minutes), take z-stack images of the head of the animals using a 40x magnification lens (Figure 2A). Discard slides after imaging.</li>
		<li>After acquisition of images, process&#160;z-stacks to obtain the maximum projection and use to quantify the number of foci in neurons located on the nerve ring area. Foci are brighter punctuated signals that can be differentiated from the dimmer soluble signal in the background. 
		NOTE: We specifically selected the nerve ring area to quantify rgef-1p::polyQ::YFP aggregates since this is the region where most neurons converge to make synaptic connections. However, in order to measure levels of aggregate formation in motor neurons, rgef-1p::polyQ::YFP aggregates located on the dorsal or ventral nerve cord can be quantified.</li>
		<li>Plot the progression of YFP foci accumulation from D4, D6, D8 and D10. Do this on an XY graph where X represents days of adulthood and Y represents number of rgef-1p::polyQ::YFP foci (Figure 2B). The number of foci in the experimental condition (e.g., gene downregulation or overexpression) is compared to control animals. Statistical analysis of foci counts for neurons is done in the same manner as for muscle polyQ foci; see steps 4.1.4&#160;and the associated notes.</li>
	</ol>
	</li>
	<li>Measuring body bends as a surrogate measure of polyglutamine proteotoxicity in neurons. 
	NOTE: The proteotoxic stress induced from the progressive aggregate accumulation of neuronal polyQ is determined by looking at motor neuron defects through a thrashing assay. Quantify the number of body bends in a period of 30 seconds while suspended in M9 physiological buffer (Supplemental File 1).
	<ol>
		<li>On Day 2 of adulthood, pick 10 synchronized animals from the plate and place on a 10 &#956;L drop of M9 buffer on a microscope slide. Repeat this step at least four times to get a sample of 40 or more animals.</li>
		<li>Video record the movement of the 10 animals placed on the microscope slide with 10 &#956;L of M9 buffer for a period of 30 s on a stereomicroscope with a video-capable camera. Repeat this step 4 times for a total sample number of 40. Zoom and position should be adjusted such that all the animals are visible in the frame for the full 30 seconds.</li>
		<li>Once all the videos with the animals to be analyzed are recorded, play the video and score the body bends of each animal. One body bend is defined as when the vulva of the animal goes from one side to the opposite and all the way back to the starting position. 
		NOTE: We find wild type animals typically have 49 &#177; 0.79 body bends in 30 seconds.</li>
		<li>Plot the number of body bends for each animal in a column graph where each dot represents the number of body bends in 30 s (Y axis) with the different conditions tested on the X axis. The number of body bends of unc-54p::polyQ::YFP animals is compared to control animals (Figure 2C). Perform statistical analysis between the groups independently for each trial; if the assay is repeated at multiple time points, analyze the data independently for each time point.
		<ol>
			<li>When considering only two conditions, perform an independent-sample t-test.</li>
			<li>When considering more than two conditions at once, first perform an omnibus one-way ANOVA analysis, including the data for all the conditions at that time trial/time point. If the omnibus ANOVA yields a significant p-value (i.e., p &#60; 0.005), perform a post-hoc analysis with pairwise independent sample t-tests to determine the specific conditions between which a significant difference in body bend count was detected.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

The authors declare that they have no competing financial interests.

Aging is characterized by a gradual decline in proteostasis. Proteostasis is maintained by a complex system, the proteostatic network, for the coordinated, dynamic, stress-responsive control of protein folding, degradation, and translation. Why proteostasis fails in the course of aging is poorly understood, but a decaying epigenome, declining inducibility of stress responses, and loss of compensatory crosstalk all coincide with this breakdown. In C. elegans, the transcriptional inducibility of multiple forms of ...

Protein homeostasis (proteostasis) is defined as the cellular ability to maintain proper function and folding of the proteome. The inherent challenge to proteostasis is ensuring all proteins are properly folded and maintained in a native conformation, which is further amplified by the varied nature of protein size, amino acid composition, structural conformation, stability, turnover, expression, sub-cellular compartmentalization, and modifications1. Proteostasis is maintained through the coordinated action of a large proteostatic network, consisting of approximately 2000 unique proteins, which regulate proper synthesis, folding, trafficking, and degradation within the proteome2,3. The workhorse components of the proteostatic network are nine major families of molecular chaperones4. Every tissue and cell type preferentially utilizes specific subsets of molecular chaperones, presumably in alignment with the differing demands of distinct proteomes5.
One hallmark of normal organismal aging is the progressive decline and collapse of cellular proteostasis, which is thought to be an underlying basis for the onset and progression of a growing number of age-associated diseases. For instance, Alzheimer&#8217;s disease, Parkinson&#8217;s disease, Huntington&#8217;s disease, and Amyotrophic Lateral Sclerosis (ALS) share a common characteristic: in each case manifestation of neurodegeneration is driven by genetic alterations that predisposes a mutant protein to aggregation (amyloid-&#946;/Tau, &#945;-synuclein, HTT, FUS/TBD-43/SOD-1, respectively)6,7,8,9,10. During aging, the integrity and inducibility of the proteostatic network declines, which results in the accumulation of proteotoxic aggregates that result in cellular dysfunction and neurodegeneration. Of note, protein conformational diseases are not unique to neurons, and occur across multiple tissues, as highlighted by type II diabetes, multiple myeloma, and cystic fibrosis11,12,13,14. Therefore, elucidating mechanisms capable of preserving proteostasis will facilitate the development of targeted interventions for the treatment of disease and to promote healthy aging.
The small soil nematode Caenorhabditis elegans (C. elegans) has been instrumental in discovering genes and elucidating pathways that alter proteostasis. Many components of the proteostatic network and the signal transduction pathways that regulate proteostasis are evolutionarily conserved. Furthermore, C. elegans has reduced complexity and redundancy relative to vertebrate systems, making it more amenable to genetic analysis and gene discovery. Additional advantages of C. elegans that have led to it being widely used as a model system to study proteostasis include: powerful genetic and functional genomics, a short life cycle (3 days) and lifespan (3 weeks), a compact and well-annotated genome, the availability of a wide assortment of genetic mutants, and the ease of visualizing tissue-specific changes in cell biology using fluorescent reporters.
The progressive decay of proteostasis during aging can be easily quantified in C. elegans. The Morimoto laboratory first demonstrated that a polyglutamine expansion fused to yellow fluorescent protein (polyQ::YFP) could be used to quantify proteostatic decline in C. elegans during aging15,16,17,18. YFP fusions to 35 glutamine repeats or more result in an age-associated formation of fluorescent foci along with signs of cellular pathology. Of note, this range of glutamine expansion mirrors the length of the polyglutamine tract of the Huntingtin protein at which Huntington&#8217;s Disease pathology begins to be observed in humans (typically &#62;35 CAG repeats)19. Strains with expression of polyQ::YFP within muscle, intestinal, or neuronal cells have been utilized to confirm that the age-associated decline of proteostasis occurs across different cell and tissue types. Muscle-specific polyQ::YFP expression (i.e., unc-54p::Q35::YFP) has been the most widely used tissue-specific reporter, as accumulating fluorescent foci are easy to quantify over the first few days of adulthood using a simple fluorescent dissecting microscope (Figure 1A-1B). Additionally, animals become paralyzed during mid-life, as the proteome within the muscle collapses due to the proteotoxic effect of the reporter (Figure 1C). Similarly, the age-associated decline in neuronal proteostasis can be followed (rgef-1p::Q40::YFP) by directly quantifying foci/aggregate formation and age-associated declines in coordinated body-bends after placing animals into liquid (Figure 2).
Here, we present a detailed protocol on how to measure the age-dependent progression of protein aggregate accumulation and the associated proteotoxicity induced by the expression of polyglutamine repeats within neuronal and muscle tissue in C. elegans. We provide examples of typical results generated using these strains and methods. Further, we show how we have utilized these methods to study transcriptional regulation of the proteostatic network. We discuss additional ways these reporters can be easily integrated with other existing reagents or adapted for larger screens.

<table><tbody><tr><td>24 Well Culture Plates</td><td>Greiner Bio-One</td><td>#662102</td><td></td></tr><tr><td>2 mL 96-well plates</td><td>Greiner Bio-One</td><td>#780286</td><td></td></tr><tr><td>600 &amp;micro;L 96-well plates</td><td>Greiner Bio-One</td><td>#786261</td><td></td></tr><tr><td>96-pin plate replicator</td><td>Nunc</td><td>250520</td><td></td></tr><tr><td>Air-permeable plate seal</td><td>VWR</td><td>60941-086</td><td></td></tr><tr><td>bacteriological agar</td><td>Affymetrix/USB</td><td>10906</td><td></td></tr><tr><td>bacto-peptone</td><td>VWR</td><td>90000-368</td><td></td></tr><tr><td>C. elegans RNAi clone library in HT115 bacteria- Ahringer</td><td>Source Bioscience</td><td>C. elegans RNAi Collection (Ahringer)</td><td>See also Kamath et. al, Nature 2003.</td></tr><tr><td>C. elegans RNAi clone library in HT115 bacteria- Vidal</td><td>Source Bioscience</td><td>C. elegans ORF-RNAi Resource (Vidal)</td><td>See also Rual et. al, Genome Research 2004. This library is also available from Dharmacon.</td></tr><tr><td>FuDR (5-Fluoro-2&#039;-deoxyuridine)</td><td>Alfa Aesar</td><td>L16497</td><td></td></tr><tr><td>Glass microscope cover slips</td><td>VWR</td><td>48404-455</td><td></td></tr><tr><td>Glass microscope slides</td><td>VWR</td><td>160004-422</td><td></td></tr><tr><td>IPTG (isopropyl beta-D-1-thigalactopyranoside)</td><td>Gold Bio</td><td>12481C100</td><td></td></tr><tr><td>Retangular non-treated single-well plate, 128x86mm</td><td>Thermo-Fisher</td><td>242811</td><td></td></tr><tr><td>Sodium Azide, CAS #26628-22-8</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td>Zeiss Axio Imager M2m microscope with AxioVision v4.8.2.0 software</td><td>Zeiss</td><td>unknown</td><td></td></tr><tr><td>Zeiss StemiSV11 M2 Bio Quad microscope</td><td>Zeiss</td><td>unknown</td><td></td></tr></tbody></table>

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.

In C. elegans, the polyglutamine repeat model has been instrumental for the identification of genes that regulate the proteostatic network. For instance, we previously showed that the homeodomain interacting protein kinase (hpk-1), a transcriptional cofactor, influences proteostasis during aging by regulating expression of autophagy and molecular chaperones31. We found that loss of hpk-1, either by RNAi silencing or in hpk-1(pk1393) null mutant animals, increase...

Watch this Scientific Journal Video about Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans at JoVE.com

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.

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 ...

quantifying-tissue-specific-proteostatic-decline-caenorhabditis

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JoVE Journal

Biology

2.8K Views.  University of Rochester Medical Center. 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.

Video: Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

Genetics

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

Caenorhabditis elegans serve as an excellent model system with robust and low-cost methods for surveying healthspan, lifespan, and resilience to stress.

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many ...

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

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million people worldwide. Since many details about PD etiology remain unknown, studies investigating genetic and environmental contributors to PD are needed to discover methods of prevention, management, and treatment. Proper characterization of dopaminergic neuronal loss may be relevant not only to PD research, but to other increasingly prevalent neurodegenerative disorders.
There are established genetic and chemical models of dopaminergic neurodegeneration in the Caenorhabditis elegans model system, with easy visualization of neurobiology supported by the nematodes&#39; transparency and invariant neuronal architecture. In particular, hermaphroditic C. elegans&#39; dopaminergic neuron morphological changes can be visualized using strains with fluorescent reporters driven by cell-specific promotors such as the dat-1 dopamine transporter gene, which is expressed exclusively in their eight dopaminergic neurons.
With the capabilities of this model system and the appropriate technology, many laboratories have studied dopaminergic neurodegeneration. However, there is little consistency in the way the data is analyzed and much of the present literature uses binary scoring analyses that capture the presence of degeneration but not the full details of the progression of neuron loss. Here, we introduce a universal scoring system to assess morphological changes and degeneration in C. elegans&#39; cephalic neuron dendrites. This seven-point scale allows for analysis across a full range of dendrite morphology, ranging from healthy neurons to complete dendrite loss, and considering morphological details including kinks, branching, blebs, and breaks. With this scoring system, researchers can quantify subtle age-related changes as well as more dramatic chemical-induced changes. Finally, we provide a practice set of images with commentary that can be used to train, calibrate, and assess the scoring consistency of researchers new to this method. This should improve within- and between- laboratory consistency, increasing rigor and reproducibility.

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

In this article, we showcase how to use a seven-point scoring system to consistently quantify changes to dopaminergic neuron dendrite morphology in C. elegans. This system is intended for analyses of dopaminergic neurodegeneration assays utilizing genetic, chemical, and age-based models of neurodegenerative disorders.

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million ...

Neuroscience

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and degradation instigates a multitude of age-related diseases. Decline of proteostasis quality control mechanisms is a hallmark of ageing. Biochemical methods to detect de novo protein synthesis are still limited, have several disadvantages and cannot be performed in live cells or animals. Caenorhabditis elegans, being transparent and easily genetically modified, is an excellent model to monitor protein synthesis rates by using imaging techniques. Here, we introduce and describe a method to measure de novo protein synthesis in vivo utilizing fluorescence recovery after photobleaching (FRAP). Transgenic animals expressing fluorescent proteins in specific cells or tissues are irradiated by a powerful light source resulting in fluorescence photobleaching. In turn, assessment of fluorescence recovery signifies new protein synthesis in cells and/or tissues of interest. Hence, the combination of transgenic nematodes, genetic and/or pharmacological interventions together with live imaging of protein synthesis rates can shed light on mechanisms mediating age-dependent proteostasis collapse.

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Here, we introduce and describe a nonradioactive and noninvasive method to assess de novo protein synthesis in vivo, utilizing the nematode Caenorhabditis elegans and fluorescence recovery after photobleaching (FRAP). This method can be combined with genetic and/or pharmacological screens to identify novel modulators of protein synthesis.

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and ...

Quantification of Insoluble Protein Aggregation in Caenorhabditis elegans during Aging with a Novel Data-Independent Acquisition Workflow

We and others have shown that the aging process results in a proteome-wide accumulation of insoluble proteins. Knocking down genes encoding the insoluble proteins over 40% of the time results in an extension of the lifespan in C. elegans, suggesting that many of these proteins are key determinants of the aging process. Isolation and quantitative identification of these insoluble proteins are crucial to understand key biological processes that occur during aging. Here, we present a modified and improved protocol that details how to extract and isolate the SDS-insoluble proteins (insolublome) from C. elegans more efficiently to streamline mass spectrometric workflows via a novel label-free quantitative proteomics analysis. This improved protocol utilizes a highly efficient sonicator for worm lysis that greatly increases efficiency for protein extraction and allows us to use significantly less starting material (approximately 3,000 worms) than in previous protocols (typically using at least 40,000 worms). Subsequent quantitative proteomic analysis of the insolublome was performed using data-dependent acquisition (DDA) for protein discovery and identification and data-independent acquisition (DIA) for comprehensive and more accurate protein quantification. Bioinformatic analysis of quantified proteins provides potential candidates that can be easily followed up with other molecular methods in C. elegans. With this workflow, we routinely identify more than 1000 proteins and quantify more than 500 proteins. This new protocol enables efficient compound screening with C. elegans. Here, we validated and applied this improved protocol to wild-type C. elegans N2-Bristol strain and confirmed that aged day-10 N2 worms showed greater accumulation of the insolublome than day-2 young worms.

Quantification of Insoluble Protein Aggregation in Caenorhabditis elegans during Aging with a Novel Data-Independent Acquisition Workflow

This novel workflow efficiently extracts and isolates SDS-insoluble proteins (insolublome) from Caenorhabditis elegans with minimal starting material for quantitative differential proteomic analysis. The protocol uses a comprehensive data-independent acquisition mass spectrometry analysis to quantify the insolublome and bioinformatic analysis to gain biological insights into aging mechanisms and pathologies.

We and others have shown that the aging process results in a proteome-wide accumulation of insoluble proteins. Knocking down genes encoding the insoluble ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

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Chapters in this video

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

Measuring Caenorhabditis elegans Life Span on Solid Media

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

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

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Quantification of Insoluble Protein Aggregation in Caenorhabditis elegans during Aging with a Novel Data-Independent Acquisition Workflow