This work was supported by funding from the DZNE and a Marie Curie International Reintegration Grant (322120 to D.C.D.)

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The authors have nothing to disclose.

Here we report a methodology to isolate highly-insoluble protein aggregates from aging C. elegans subjected to RNAi for analysis by mass spectrometry and Western blotting. We show that improving proteostasis by reducing IIS greatly prevents age-dependent protein aggregation. By selecting specific aggregation-prone proteins to overexpress in C. elegans, it is possible to dissect further the mechanisms modulating inherent protein aggregation.
Inherent Age-dependent Protein Aggregation Versus Disease-associated Protein Aggregation 
This current work is based on the previous findings that protein aggregation is not restricted to specific proteins aggregating in a disease context. By focusing on endogenous aggregation-prone proteins rather than ectopically expressed disease-associated proteins, the expectation is to provide novel insight into physiological regulation of the aggregation process. As age-dependent protein aggregates are found in different cellular locations5, their study should also provide insight into compartment specific quality-control mechanisms. Overall, the mechanisms that control inherent protein aggregation could also be relevant in preventing disease protein aggregation. Of note, although age-dependent protein aggregation resembles in several aspects disease protein aggregation, it still remains unclear whether these aggregates contain amyloid fibrils, a characteristic of many disease aggregates.
Choice of C. Elegans Model: Advantages and Limitations 
Compared to a mammalian model, one of the greatest advantages of using C. elegans to study an aging-related characteristic is its very short lifespan. Several hundred proteins consistently become highly-aggregation-prone during aging and this large increase in insolubility is easily detectable even with a simplified biochemical extraction. However, a major limitation of using C. elegans for studying age-dependent protein aggregation is the necessity to isolate large numbers of aged animals. As C. elegans are hermaphrodites and produce approximately 220 or more progeny during their reproductive phase19, it is essential to prevent reproduction or eliminate progeny in order to age a population. Several options are available to induce sterility: One option is to use the chemical fluorodeoxyuridine (FUdR), an inhibitor of DNA synthesis. However, FUdR leads to numerous side-effects including changes in proteostasis and reduced protein aggregation20,21,22. Importantly, FUdR induces genotype-specific responses23. Another option is to use temperature-sensitive sterile mutants. For example, spe-9(hc88), fer-15(b26), fem-1(hc17), glp-1(e2141), and gon-2(q388) have been used previously for mass spectrometry analyses of the age-dependent aggregating proteome5,8.
Yet there are several limitations associated with using these mutants. First, as the sterility is temperature-dependent, it is necessary to grow the animals at 25 &#176;C, which constitutes a mild stress and accelerates aging as well as protein aggregation. Conversely, we have observed that gon-2, glp-1 and fem-1 mutants are longer-lived compared to wild-type when maintained at 25 &#176;C (data not shown). Second, there are specific limitations associated with each type of reproductive defect. One disadvantage of using sperm-defective mutants is the production of unfertilized oocytes (also produced in aged wild-type hermaphrodites). The quality of these oocytes greatly declines with age and they accumulate in the gonad24,25. Quantitative mass spectrometry analysis demonstrates that sperm-defective mutants have several fold higher levels of insoluble proteins accumulating with age compared to gonad-less or germline-deficient mutants. This indicates protein misfolding and aggregation located in the gonad5, in agreement with previous studies12,13 and in vivo experiments revealing aggregated proteins located in the abnormal oocyte clusters5. Therefore, excessive protein aggregation in the gonad would mask more subtle changes in protein aggregation in the somatic tissues. This should also be considered when comparing interventions that act to different extents on reproductive and somatic tissue proteostasis. Therefore, to assess only somatic protein aggregation, we favor using gonad-less gon-2 mutants. An alternative would be to use germline-deficient glp-1 mutants. However, we note that these animals have less inherent protein aggregation with age compared to gon-2 mutants5. This is likely a consequence of improved proteostasis in somatic tissues due to signaling from the germline-defective gonad26,27,28. Another alternative is to use wild-type animals and remove progeny every day by sedimentation. However, this is problematic due to the difficulty of completely removing all progeny.
Another general limitation of using C. elegans is its small size which makes specific tissue extractions challenging. As such whole animal extractions do not provide relevant information related to how different conditions modulate protein aggregation in specific tissues. Of note, this issue could potentially be circumvented by using a recently developed method based on fluorescence-activated cell sorting to isolate specific cells from C. elegans29. Yet it remains to be determined whether this method would be suitable to obtain sufficient material for insoluble protein extraction and whether it would be applicable to aged animals. Alternatively, tissue specific protein aggregation and its regulation can be investigated by additional in vivo experiments. As transgenic animals expressing a fluorescently-labeled aggregation-prone protein of choice can be rapidly generated and as the animals are transparent, it is relatively easy to examine protein aggregation in situ.
Overall, it is essential to follow up the proteomic results with in vivo experiments in a wild-type background. The combination of a biochemical and microscopy-based characterization enables a comprehensive study of protein aggregation and an assessment of the role of genes of interest. The latter is facilitated in C. elegans by RNAi mediated through feeding and the availability of whole genome RNAi libraries. In addition, a large number of characterized mutants exist to confirm results obtained by RNAi or to use instead of RNAi.
Liquid Culture and Biochemical Extraction: Advantages and Limitations 
As highly-aggregated proteins make up only a small fraction of total proteins, it is necessary to start the extraction with a large number of animals. With large quantities of insoluble proteins, it is then feasible to perform extensive peptide fractionation to reduce sample complexity for mass spectrometry and thus improve identification of lower abundant proteins. If only small amounts of insoluble proteins are required, an alternative is to grow worms on plates and homogenize them with ceramic beads. One of the disadvantages of liquid culture is that in the case of a contamination, the whole culture is affected and must be discarded. In general, C. elegans aging is strongly influenced by temperature and this parameter must be tightly controlled in the liquid culture to avoid variations in age-dependent protein aggregation. Temperature control is also essential to obtain a homogenous population of gonad-less animals when using the gon-2(-) mutants.
Depending on the goal of the study, it is important to consider different extraction methods. The biochemical extraction presented here was initially adapted from protocols to isolate disease-associated aggregates11,30,31. We chose relatively high concentrations of detergents such as 0.5% SDS to isolate the more insoluble aggregates. Also by pelleting the 0.5% SDS insoluble aggregates with low centrifugal speeds, this protocol would only isolate the larger aggregates. Alternatively, a less stringent protocol was recently published10. In this study, more soluble and smaller aggregates were also extracted by omitting SDS and by using very high centrifugal speeds at 500,000 x g. A comparison of the different protocols shows a general increase in the number of proteins in the aggregating proteome using the less stringent protocol7. We note that long-lived animals with reduced daf-2 signaling efficiently prevented the accumulation of both SDS-soluble and SDS-insoluble aggregates in gonad-less animals7.
In Vivo Analysis of Age-Dependent Protein Aggregation 
When constructing transgenic models for following age-dependent protein aggregation, it is relevant to consider in which tissue to overexpress the aggregation-prone protein of interest and at which expression levels. We favor expression in tissues located in the head region to avoid interference with autofluorescence, which is prominent in the aging intestine. To visualize fluorescent-labeled aggregates with low-magnification, the aggregation-prone protein should be expressed at a relatively high level. On the other hand, too high expression will cause the aggregation-prone protein to form aggregates already in young animals.
Taken together, these methods will help us to understand why part of the proteome aggregates with age and ultimately lead to the development of strategies promoting healthy aging.

Protein misfolding and aggregation are recognized as a hallmark of several neurodegenerative diseases such as AD, PD, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and many others. For instance, &#945;-synuclein assemblies into amyloid fibrils that accumulate as Lewy bodies particularly in the substantia nigra of PD patients, while in ALS patients TDP-43 or FUS misfold to form cytoplasmic aggregates in degenerating motor neurons. In each of these neurodegenerative disorders, mechanisms maintaining protein homeostasis or proteostasis fail to prevent the accumulation of misfolded proteins, consequently leading to disease.
Proteostasis is critical to ensure cellular functions and under normal conditions these regulatory mechanisms tightly control the rate of protein synthesis, folding, and degradation. Several studies demonstrate that with ageing, the ability of many cells and organs to preserve protein homeostasis is gradually compromised and the physiological deterioration of the proteostasis networks with age is an important aggravating factor for neurodegenerative diseases (reviewed in references1,2,3). The fact that the protein quality control and the cellular response to unfolded protein stress are compromised with age suggests that protein misfolding and aggregation could be a general consequence of aging. Indeed, we and others have demonstrated that protein aggregation is not restricted to disease and instead part of the proteome becomes highly detergent-insoluble in aged animals4,5,6,7,8,9,10. Computational and in vivo analysis revealed that these physiological age-related aggregates resemble disease aggregates in several aspects5. The discovery of endogenous, age-dependent protein aggregation gives us the opportunity to dissect the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. At present, only limited information exists about the regulation of widespread protein insolubility and about the effects of this dysregulation on the health of the organism.
The nematode C. elegans is one of the most extensively studied model organisms in aging research as these animals have a relatively short lifespan and show many characteristic aging features observed in higher organisms. The effects of aging on protein insolubility have been studied in C. elegans by sequential biochemical fractionation based on differential solubility, which is widely used to extract disease aggregates in the field of neurodegeneration research11. By quantitative mass spectrometry, several hundred proteins were shown to become aggregation-prone in C. elegans in the absence of disease5. Here we describe in detail the protocol to grow large numbers of worms in liquid culture and the sequential extraction to isolate aggregated proteins for quantification by mass spectrometry and analysis by Western blot. Because misfolded and aggregation-prone proteins accumulate in aged C. elegans gonads and masks changes in other somatic tissues5,12,13, we use a gonad-less mutant to focus the analysis on protein insolubility in non-reproductive tissues. The method presented enables the analysis of highly-insoluble, large aggregates that are insoluble in 0.5% SDS and pelleted by relatively low centrifugal speed. Alternatively, a less stringent extraction protocol to collect also smaller and more soluble aggregates has been published elsewhere10. In addition, we describe the method used to assess aggregation in vivo in C. elegans.
Overall, these methods in combination with RNA interference (RNAi) can evaluate the role of a gene of interest in modulating age-dependent protein aggregation. For this we describe the analysis of extracts from young and aged worms with and without knockdown of a specific protein of interest using RNAi. These methods should be a powerful tool to determine which components of the proteostasis network regulate protein insolubility. Several interventions such as reduced insulin/insulin-like growth factor (IGF) 1 signaling (IIS) have been shown to dramatically delay C. elegans aging14. Longevity pathways often induce protein-quality control mechanisms and thus these pathways could be actively influencing the rate of protein aggregation. As an example, we demonstrate reduced inherent protein aggregation in long-lived animals upon inhibition of the IIS pathway7.

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			<td class="xl19" style="width:258pt" width="344">4425-2XL</td>
			<td class="xl18" style="width:125pt" width="166">Pyrex, Capacity 2,800 ml, with 3 baffle indents</td>
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			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Gray needle, 27 G x &#189; &#34;, 0.4 mm x 13 mm</td>
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			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Protease Inhibitor Cocktail</td>
			<td class="xl18" style="width:92pt" width="122">Roche</td>
			<td class="xl19">4693132001</td>
			<td class="xl16" style="width:125pt" width="166">Complete Mini EDTA-free tablets&#160;</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Octoxynol-9&#160;</td>
			<td class="xl18" style="width:92pt" width="122">Applichem</td>
			<td class="xl19">A1388</td>
			<td class="xl18" style="width:125pt" width="166">Triton X-100</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">4-Morpholineethanesulfonic acid (MES)</td>
			<td class="xl18" style="width:92pt" width="122">Sigma-Aldrich</td>
			<td class="xl19">M1317</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Nonylphenylpolyethylenglycol</td>
			<td class="xl18" style="width:92pt" width="122">Applichem</td>
			<td class="xl19">A1694</td>
			<td class="xl18" style="width:125pt" width="166">Nonidet P40 (NP40)</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">DNaseI</td>
			<td class="xl18" style="width:92pt" width="122">Roche</td>
			<td class="xl19">04716728001</td>
			<td class="xl18" style="width:125pt" width="166">recombinant, RNase free</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">RNaseA</td>
			<td class="xl18" style="width:92pt" width="122">Promega</td>
			<td class="xl19">A7973</td>
			<td class="xl18" style="width:125pt" width="166">solution</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Total protein blot staining</td>
			<td class="xl18" style="width:92pt" width="122">Thermofisher</td>
			<td class="xl19">S11791</td>
			<td class="xl18" style="width:125pt" width="166">Sypro Ruby protein blot stain</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Total protein gel staining</td>
			<td class="xl18" style="width:92pt" width="122">Thermofisher</td>
			<td class="xl19">S12001</td>
			<td class="xl18" style="width:125pt" width="166">Sypro Ruby protein gel stain</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">TCEP (tris (2-carboxyethyl) phosphine hydrochloride)</td>
			<td class="xl18" style="width:92pt" width="122">Serva</td>
			<td class="xl19">36970</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Iodoacetamide</td>
			<td class="xl18" style="width:92pt" width="122">Serva</td>
			<td class="xl19">26710</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Ammoniumbicarbonate</td>
			<td class="xl18" style="width:92pt" width="122">Sigma-Aldrich</td>
			<td class="xl19">09830</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Sequencing Grade Modified Trypsin</td>
			<td class="xl18" style="width:92pt" width="122">Promega</td>
			<td class="xl25" style="width:258pt" width="344">V5111</td>
			<td class="xl18" style="width:125pt" width="166"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Isobaric tags for relative and absolute quantitation</td>
			<td class="xl18" style="width:92pt" width="122">Sciex</td>
			<td class="xl19">4352135</td>
			<td class="xl18" style="width:125pt" width="166">iTRAQ Reagents Multiplex Kit</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Centrifuge Avanti J-26XP</td>
			<td class="xl18" style="width:92pt" width="122">Beckmann Coulter</td>
			<td class="xl19">393126</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Ultracentrifuge Optima Max-XP</td>
			<td class="xl18" style="width:92pt" width="122">Beckmann Coulter</td>
			<td class="xl19">393315</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Centrifuge 5424R</td>
			<td class="xl18" style="width:92pt" width="122">Eppendorf</td>
			<td class="xl19">5404000413</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Centrifuge 5702</td>
			<td class="xl18" style="width:92pt" width="122">Eppendorf</td>
			<td class="xl19">5702000329</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Centrifuge Megafuge 40R</td>
			<td class="xl18" style="width:92pt" width="122">Thermo Scientific</td>
			<td class="xl19">75004518</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Concentrator Plus</td>
			<td class="xl18" style="width:92pt" width="122">Eppendorf</td>
			<td class="xl19">5305000304</td>
			<td class="xl17">Centrifugal evaporator</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Fluorescent stereo-microscope M165 FC&#160;</td>
			<td class="xl18" style="width:92pt" width="122">Leica</td>
			<td class="xl19"></td>
			<td class="xl18" style="width:125pt" width="166">With Planapo 2.0x objective</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Dissection microscope</td>
			<td class="xl18" style="width:92pt" width="122">Leica&#160;</td>
			<td class="xl19"></td>
			<td class="xl17">Leica S6E</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="80" rowspan="2" style="width:161pt;height:60.0pt" width="215">High magnification microscope Zeiss Axio Observer Z1</td>
			<td class="xl18" height="80" rowspan="2" style="width:92pt;height:60.0pt" width="122">Zeiss</td>
			<td class="xl19" height="80" rowspan="2" style="width:258pt;height:60.0pt" width="344"></td>
			<td class="xl16" height="80" rowspan="2" style="width:125pt;height:60.0pt" width="166">With PlanAPOCHROMAT 20x objective and Zeiss Axio Cam MRm</td>
		</tr>
		<tr height="40" style="height:30.0pt">
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl32" height="40" style="height:30.0pt">Software</td>
			<td class="xl33" style="width:92pt" width="122"></td>
			<td class="xl32"></td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Image analysis software</td>
			<td class="xl18" style="width:92pt" width="122">ImageJ</td>
			<td class="xl19"></td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Analysis of mass spectrometry data</td>
			<td class="xl18" style="width:92pt" width="122">Protein Prospector</td>
			<td class="xl136" style="width:258pt" width="344"></td>
			<td class="xl137">http://prospector.ucsf.edu/prospector/mshome.htm</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl32" height="40" style="height:30.0pt">E.coli strain</td>
			<td class="xl33" style="width:92pt" width="122"></td>
			<td class="xl19"></td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">OP50</td>
			<td class="xl18" style="width:92pt" width="122">CGC</td>
			<td class="xl19"></td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl32" height="40" style="height:30.0pt">RNAi bacteria</td>
			<td class="xl33" style="width:92pt" width="122"></td>
			<td class="xl19"></td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">L4440</td>
			<td class="xl18" style="width:92pt" width="122">Julie Ahringer RNAi library</td>
			<td class="xl19"></td>
			<td class="xl17"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl32" height="42" rowspan="2" style="width:161pt;height:31.5pt" width="215">C. elegans mutants</td>
			<td class="xl33" height="42" rowspan="2" style="width:92pt;height:31.5pt" width="122"></td>
			<td class="xl32" height="42" rowspan="2" style="width:258pt;height:31.5pt" width="344"></td>
			<td class="xl19" height="42" rowspan="2" style="width:125pt;height:31.5pt" width="166"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl17" height="42" style="height:31.5pt">CF2253</td>
			<td class="xl18" style="width:92pt" width="122">CGC, strain name: EJ1158&#160;</td>
			<td class="xl40" style="width:258pt" width="344"></td>
			<td class="xl17">Genotype: gon-2(q388)</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl33" height="42" rowspan="2" style="width:161pt;height:31.5pt" width="215">C. elegans transgenics</td>
			<td class="xl33" height="42" rowspan="2" style="width:92pt;height:31.5pt" width="122"></td>
			<td class="xl32" height="42" rowspan="2" style="width:258pt;height:31.5pt" width="344"></td>
			<td class="xl19" height="42" rowspan="2" style="width:125pt;height:31.5pt" width="166"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl18" height="42" rowspan="2" style="width:161pt;height:31.5pt" width="215">DCD214</td>
			<td class="xl25" height="42" rowspan="2" style="width:92pt;height:31.5pt" width="122">Della David&#39;s lab at DZNE T&#252;bingen</td>
			<td class="xl40" height="42" rowspan="2" style="width:258pt;height:31.5pt" width="344"></td>
			<td class="xl68" height="42" rowspan="2" style="width:125pt;height:31.5pt" width="166">Genotype: N2; uqIs24[Pmyo-2::tagrfp::pab-1]</td>
		</tr>
		<tr height="21" style="height:15.75pt">
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="42" rowspan="2" style="width:161pt;height:31.5pt" width="215">DCD215</td>
			<td class="xl87" height="42" rowspan="2" style="width:92pt;height:31.5pt" width="122">Della David&#39;s lab at DZNE T&#252;bingen</td>
			<td class="xl40" height="42" rowspan="2" style="width:258pt;height:31.5pt" width="344"></td>
			<td class="xl68" height="42" rowspan="2" style="width:125pt;height:31.5pt" width="166">Genotype: daf-2(e1370) III; uqIs24[Pmyo-2::tagrfp::pab-1]</td>
		</tr>
		<tr height="21" style="height:15.75pt">
		</tr>
	</tbody>
</table>

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.

We used the methods presented here to evaluate how long-lived animals with reduced IIS modulate age-dependent protein aggregation. By Western blot (see step 2.2, Quick insoluble protein extraction for Western blot analysis), we analyzed the total and the insoluble protein content of young (day 3 of adulthood) and aged (day 18 of adulthood) worms on control RNAi and on RNAi targeting the insulin/IGF-1-like receptor daf-2. We observed no large changes in total protein levels with age or between control and daf-2 RNAi conditions (Figure 2A). As expected, we detected a strong age-related increase in levels of highly-insoluble proteins in control animals (Figure 2B). In comparison in the long-lived animals on daf-2 RNAi, aggregation propensity was greatly reduced. Whole protein staining of the insoluble extracts revealed a less complex protein band pattern in extracts from 18-day-old worms on daf-2 RNAi compared to age-matched controls. Quantitative mass spectrometry analysis demonstrated that long-lived animals with reduced IIS have overall less age-associated protein aggregation and importantly, daf-2 inhibition completely abrogated the age-dependent insolubility of a specific subset of proteins7. To follow-up on these findings, we focused on one of these aggregation-prone proteins, PAB-1. PAB-1 is an RNA-binding protein with a predicted &#34;prion-like&#34; domain. Antibody staining confirmed that long-lived animals maintained PAB-1 soluble with age (Figure 2C).
For in vivo analysis of PAB-1 aggregation, we produced transgenic animals expressing tagRFP::PAB-1 in the pharyngeal muscles. In young animals, tagRFP::PAB-1 was mainly diffusely located throughout the pharynx (Figure 3A, &#34;low&#34; aggregation level) but with age, we observed a progressive accumulation in bright puncta starting in the posterior pharyngeal bulb (Figure 3A, &#34;intermediate&#34; aggregation level). During aging, we also observed aggregation in the anterior pharyngeal bulb (Figure 3A, &#34;high&#34; aggregation level) in an increasing number of animals. By grouping animals into these different categories, we scored the rate of PAB-1 aggregation in a population. At day 2 of adulthood most of the worms (88%) had none or very few puncta and no animals with high aggregation levels were detected. Already at day 5, 19% of the population showed intermediate and 16% high aggregation levels, while at day 7 more than 70% of the worms presented intermediate and high aggregation levels. To analyze the effect of longevity on the age-dependent aggregation of tagRFP:PAB-1, we generated transgenic tagRFP::PAB-1 worms with a daf-2 mutation. These animals showed significantly reduced tagRFP:PAB-1 puncta formation with aging, with less than 15% of the population exhibiting intermediate and high aggregation levels even at day 7 (Figure 3B).
By using the different approaches described here, we revealed that reduced IIS prevents age-dependent protein aggregation to different extents and long-lived animals are particularly efficient in restoring the dynamics of PAB-17.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56464/56464fig1.jpg" /> 
Figure 1: Schematic of the workflow to study changes in inherent protein aggregation with age in C. elegans.&#160;Main steps are bold. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56464/56464fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56464/56464fig2.jpg" /> 
Figure 2: Protein insolubility increases with age in control animals, but not in animals with reduced daf-2 signaling. (A) Total protein staining of total fraction from day 3 and day 18 gon-2(-) mutants subjected to control and daf-2 RNAi (grown at 25 &#176;C). (B) Total protein staining of SDS-insoluble fraction from day 3 and day 18 gon-2(-) mutants subjected to control and daf-2 RNAi (grown at 25 &#176;C). Panel republished from reference7. (C) Immunoblot detecting PAB-1 in SDS-insoluble fraction, relevant protein bands indicated by red arrow. Panel republished from reference7. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56464/56464fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56464/56464fig3.jpg" /> 
Figure 3: Reduced daf-2 signaling prevents the accumulation of tagRFP::PAB-1 puncta with age. (A) Representative pictures of worms with tagRFP::PAB-1 overexpression in the pharyngeal muscles (high magnification microscope, tagRFP detection settings: excitation 558 nm, emission 583 nm). Animals with low (0-10 tagRFP::PAB-1 puncta in posterior pharyngeal bulb), intermediate (more than 10 puncta in the posterior pharyngeal bulb), or high aggregation levels (more than 10 puncta in the anterior pharyngeal bulb) are shown. Scale bar = 20 &#181;m. (B) Quantification of different tagRFP::PAB-1 aggregation levels with age in wildtype and daf-2 mutant background reveals strongly delayed tagRFP::PAB-1 aggregation with age in long-lived animals. Day 2, day 5, and day 7 daf-2(-) versus wildtype background, low compared to intermediate and high aggregation levels: Fisher&#39;s exact test, ****p &#60;0.0001. Panel republished from reference7. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56464/56464fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Stock solution</td>
			<td>Amount</td>
			<td>Final concentration</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td colspan="4">Liquid culture, total volume 300 mL</td>
		</tr>
		<tr>
			<td>S basal (For 500 mL: 5.9 g NaCl, 50 mL 1 M Potassium phosphate pH 6)</td>
			<td>200 mL</td>
			<td></td>
			<td>1 M Potassium phosphate pH 6: For 1 L: 129 g KH2PO4 monobasic, 52 g K2HPO4 dibasic anhydrous</td>
		</tr>
		<tr>
			<td>1 M Potassium citrate pH 6 (For 400 mL: 13.1 g citric acid monohydrate, 134.4 g tri-potassium citrate monohydrate)</td>
			<td>3 mL</td>
			<td>10 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>Trace metals solution (For 1 L: 1.86 g Ethylenediaminetetraacetic acid (EDTA), 0.69 g FeSO4 &#183; 7 H2O, 0.2 g MnCl2 &#183; 4 H2O, 0.29 g ZnSO4 &#183; 7 H2O, 0.025 g CuSO4 &#183; 5 H2O)</td>
			<td>3 mL</td>
			<td></td>
			<td>Store stock solution in the dark at 4 &#176;C</td>
		</tr>
		<tr>
			<td>1 M MgSO4 (For 500 mL: 123.3 g)</td>
			<td>900 &#181;L</td>
			<td>3 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M CaCl2 (For 500 mL: 73.5 g)</td>
			<td>900 &#181;L</td>
			<td>3 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;g/mL Carbendazim (For 50 mL: 10 mg in Methanol)</td>
			<td>150 &#181;L</td>
			<td>100 ng/mL</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mg/mL Cholesterol (For 100 mL: 0.5 g in Ethanol)</td>
			<td>300 &#181;L</td>
			<td>5 &#181;g/mL</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Reassembly (RAB) high-salt extraction buffer, 30 mL</td>
		</tr>
		<tr>
			<td>1 M 4-Morpholineethanesulfonic acid (MES)</td>
			<td>3 mL</td>
			<td>100 mM</td>
			<td>RAB stock buffer component&#160;</td>
		</tr>
		<tr>
			<td>0.5 M Ethyleneglycoltetraacetic acid (EGTA)</td>
			<td>60 &#181;L</td>
			<td>1 mM</td>
			<td>RAB stock buffer component&#160;</td>
		</tr>
		<tr>
			<td>0.5 M Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>6 &#181;L</td>
			<td>0.1 mM</td>
			<td>RAB stock buffer component&#160;</td>
		</tr>
		<tr>
			<td>1 M MgSO4</td>
			<td>15 &#181;L</td>
			<td>0.5 mM</td>
			<td>RAB stock buffer component&#160;</td>
		</tr>
		<tr>
			<td>5 M NaCl</td>
			<td>4.5 mL</td>
			<td>750 mM</td>
			<td>RAB stock buffer component&#160;</td>
		</tr>
		<tr>
			<td>1 M Sodium fluoride (NaF)</td>
			<td>600 &#181;L</td>
			<td>20 mM</td>
			<td>RAB stock buffer component&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Complete to 30 mL total with ddH2O</td>
		</tr>
		<tr>
			<td>50x Protease Inhibitor Cocktail</td>
			<td>*</td>
			<td>2x</td>
			<td rowspan="4">*Add reagents after taking the amount of RAB stock buffer that is needed (for the extraction for mass spectrometry 6 mL).</td>
		</tr>
		<tr>
			<td>100 mM Phenylmethylsulfonyl fluoride (PMSF) in isopropanol, -20&#176;C</td>
			<td>*</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>10 U/&#181;L DNaseI</td>
			<td>*</td>
			<td>200 U/mL</td>
		</tr>
		<tr>
			<td>4 mg/mL RNaseA</td>
			<td>*</td>
			<td>100 &#181;g/mL&#160;</td>
		</tr>
		<tr>
			<td colspan="4">RAB with 1 M sucrose, 30 mL</td>
		</tr>
		<tr>
			<td>2 M sucrose (For 50 mL: 34.2 g)</td>
			<td>15 mL</td>
			<td>1 M</td>
			<td>Add the RAB buffer reagents besides DNaseI and RNaseA</td>
		</tr>
		<tr>
			<td colspan="4">Radioimmunoprecipitation assay (RIPA) buffer, 30 mL</td>
		</tr>
		<tr>
			<td>1 M Tris(hydroxymethyl)aminomethane (Tris) pH 8</td>
			<td>1.5 mL</td>
			<td>50 mM</td>
			<td>RIPA stock buffer component</td>
		</tr>
		<tr>
			<td>5 M NaCl</td>
			<td>0.9 mL</td>
			<td>150 mM</td>
			<td>RIPA stock buffer component</td>
		</tr>
		<tr>
			<td>0.5 M EDTA</td>
			<td>0.3 mL</td>
			<td>5 mM</td>
			<td>RIPA stock buffer component</td>
		</tr>
		<tr>
			<td>10 % Sodium dodecyl sulfate (SDS)</td>
			<td>1.5 mL</td>
			<td>0.50%</td>
			<td>RIPA stock buffer component</td>
		</tr>
		<tr>
			<td>10 % Sodium deoxycholate (SDO)</td>
			<td>1.5 mL</td>
			<td>0.50%</td>
			<td>RIPA stock buffer component</td>
		</tr>
		<tr>
			<td>Nonylphenylpolyethylenglycol (NP40)</td>
			<td>0.3 mL</td>
			<td>1%</td>
			<td>RIPA stock buffer component</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Complete to 30 mL total with ddH2O</td>
		</tr>
		<tr>
			<td>100 mM PMSF</td>
			<td>*</td>
			<td>1 mM</td>
			<td rowspan="2">*Add reagents after taking the amount of RIPA stock buffer that is needed (for the extraction for mass spectrometry 10 mL).</td>
		</tr>
		<tr>
			<td>50x Protease Inhibitor Cocktail</td>
			<td>*</td>
			<td>1x</td>
		</tr>
		<tr>
			<td colspan="4">Urea/SDS buffer, 10 mL</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>4.8 g</td>
			<td>8 M</td>
			<td></td>
		</tr>
		<tr>
			<td>10 % SDS</td>
			<td>2 mL</td>
			<td>2%</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>77 mg</td>
			<td>50 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Tris pH 8</td>
			<td>0.5 mL</td>
			<td>50 mM&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Complete to 10 mL total with ddH2O</td>
		</tr>
		<tr>
			<td colspan="4">Dialysis buffer, 1 L</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>6 g</td>
			<td>50 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>154 mg</td>
			<td>1 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mM PMSF</td>
			<td>1 mL</td>
			<td>0.1 mM</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Adjust to pH 7.5 and complete to 1 L total with ddH2O</td>
		</tr>
	</tbody>
</table>
Table 1: Buffers for liquid culture, insoluble protein extraction, and dialysis.

Watch this Scientific Journal Video about Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans at JoVE.com

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.

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 ...

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8.4K Views.  German Center for Neurodegenerative Diseases (DZNE). 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.

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

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

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 ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

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

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

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

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