Name: quantifying tissue-specific proteostatic decline in caenorhabditis elegans
Uploaded: 2021-09-07T13:00:00
Description: Watch this Scientific Journal Video about Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans at JoVE.com

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 stu...

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 &#181;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&#39;-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 ...

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