Name: methods to study changes in inherent protein aggregation with age in caenorhabditis elegans
Uploaded: 2017-11-26T13:00:00
Description: Watch this Scientific Journal Video about Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans at JoVE.com

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

NOTE: For a better understanding of the procedure, a schematic of the workflow (Figure 1) is attached.
1. Growth of Large Numbers of Young and Aged C. elegans Subjected to RNAi Targeting a Gene of Interest
NOTE: Use C. elegans temperature-induced sterile gon-2(q388) mutants (CF2253) to obtain large aged-synchronized populations. During all steps, it is important to work under semi-sterile conditions with an open...

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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 Prot...

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="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 a...

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 ...

The overall goal of this experiment is to detect changes in protein insolubility with age in C.elegans and to evaluate how targeted gene knockdown influences this aging marker. This method can help answer key questions in the field of aging and proteostasis such as why organisms fail to maintain a healthy proteome with age. The main advantage of this technique is the opportunity to study protein aggregation during normal aging in the absence of disease processes.Though this method can provide insight into protein aggregation in C.elegans, it can also be adapted to other model organisms, for example to study age-related protein insolubility in mouse. To begin treatment, add 207.45 milliliters of S Basal media with additional reagents as described in the accompanying text protocol to a 2, 800 milliliter Fernbach culture flask. Add a final concentration of 50 micrograms per milliliter of Carbenicillin and one millimolar of IPTG and close the flask with a membrane screw cap.Take the L1's out of the 25 degree Celsius incubator and transfer them to 15 milliliter tubes. Centrifuge the L1's at 1, 900 times g for three minutes. After spinning, remove the supernatant.Under a microscope, count the L1's per two microliters and average the numbers obtained from at least nine drops. Next, add 50, 000 worms for the young worm collection and 100, 000 worms for the aged worm collection into four Fernbach culture flasks prepared in the previous step. Then, add control RNAi bacteria and RNAi bacteria for the gene of interest proportionally to the number of worms.After adding bacteria, complete worm culture with S Basal to bring the total volume to 300 milliliters. Incubate the worm culture at 25 degrees Celsius in a shaking incubator with 150 rpm until collection. Collect young worms at day two or three to measure basal levels of protein solubility.Pour the worms from one flask into a separation funnel and let the worms sediment for 10 minutes at room temperature. After the worms sediment, open the stop cock and drip the worms into one 50 milliliter tube. Next, split the worm pellet into two 15 milliliter tubes.Fill up the tubes to 15 milliliters with M9 and centrifuge the worms. To wash the worms, remove the supernatant and fill the tube up to 15 milliliters with M9.Repeat the centrifugation. Once washing is complete, transfer the worms to two 50 milliliter tubes and use ice cold M9 to fill up the total volume to 20 milliliters.To remove bacteria and dead worms, add the two 20 milliliter diluted worm pellets to two 50 milliliter tubes filled with 20 milliliters of ice cold 60%sucrose. Quickly centrifuge the tubes. With a 25 milliliter pipette, carefully remove up to 15 milliliters of the top worm layer.Place the top worm layer directly into 37 milliliters of M9 plus octoxynol-9 prepared on ice. Then, centrifuge the tubes at 2, 700 times g for three minutes at four degrees Celsius acceleration nine and deceleration seven. After discarding the supernatant, transfer the pellet into four 15 milliliter tubes and wash them twice with ice cold M9 plus octoxynol-9.Then, centrifuge the tubes. Remove the supernatant and fill the tubes up to the 15 milliliter mark with ice cold M9 plus octoxynol-9. Wash the worms with ice cold M9 and combine the four tubes into two tubes.Fill up the tubes to 15 milliliters and centrifuge. Remove the supernatant, fill up the two tubes with M9 at room temperature to a total volume of four milliliters and rotate them on a nutating mixer at 25 degrees Celsius for 40 minutes. After nutating, wash the worms twice with ice cold M9 plus octoxynol-9.Then, wash them twice with M9 and transfer the worms to one tube. Wash the worms in the reassembly or RAB high salt extraction buffer without inhibitors before collection. Remove the supernatant until there is no liquid on top of the worm pellet.Then, estimate the volume of the worm pellet and add an identical volume of RAB with inhibitors. Prepare a 50 milliliter tube half filled with liquid nitrogen on dry ice and using a Pasteur pipette, draw up the worms and slowly drip them into the tube. Allow the liquid nitrogen to evaporate and store the frozen worms at minus 80 degrees Celsius until further processing.Cool down the mortar with liquid nitrogen and perform the animal disruption on dry ice. Transfer the frozen worms to the pre-cooled mortar and grind them for 2.5 minutes. Add 100 milliliters of liquid nitrogen to the powder and grind for another 2.5 minutes.Use a microscope to check that the worm bodies are broken into small pieces. Then, transfer the powder into two milliliter tubes and store them at minus 80 degrees Celsius. On dry ice, weigh out two times 350 milligrams of ground worms per time point and per RNAi bacteria into two milliliter tubes.To remove the high salt soluble proteins, add two volumes per weight of already prepared RAB with inhibitors, one millimolar PMSF, 200 units per milliliter DNase I, and 100 micrograms per milliliter RNase A to each tube and solubilize the powder on ice. Draw up the suspension into a one milliliter syringe 15 times and incubate it on ice for 10 minutes. Centrifuge at 18, 400 times g for 20 minutes at four degrees Celsius.Go through the fat layer and collect the supernatant containing high salt soluble proteins into one two milliliter tube per condition. Remove the fat and discard it. Aliquot high salt soluble proteins to freeze at minus 80 degrees Celsius.To discard lipids, solubilize the pellet with 700 microliters of RAB with inhibitors containing one molar sucrose without DNase I and RNase A.Draw up the suspension into a syringe 10 times and incubate it on ice for five minutes. Be sure to remove all of the supernatant and lipids and discard them. To remove SDS-soluble proteins, solubilize the pellet with 700 microliters of RIPA buffer.Draw up the suspension into a syringe 10 times and incubate it for 10 minutes on ice. Centrifuge at 18, 400 g for 20 minutes at four degrees Celsius. Collect the supernatant containing SDS-soluble proteins and aliquot for freezing at minus 80 degrees Celsius.Pool two samples of each condition together after solubilizing each pellet with 500 microliters of RIPA buffer and draw up the suspension into a syringe 10 times. Be careful to remove all of the supernatant and discard it. Solubilize the final pellet containing highly insoluble proteins with 400 microliters 70%formic acid and draw up the suspension into a syringe 20 times.Incubate the suspension for 20 minutes on ice. Centrifuge the suspension in ultracentrifuge tubes to remove worm cuticle debris. Collect the supernatant that contains highly insoluble proteins and freeze it at minus 80 degrees Celsius.Total protein staining of insoluble protein content of young and aged worms revealed a strong age-related increase in highly insoluble protein levels in control animals and not in long lived animals. Antibody staining of PAB-1, an RNA binding protein with a predicted prion-like domain, confirmed mass spectrometry data that long lived animals maintained PAB-1 soluble with age. In vivo analysis was performed of transgenic animals expressing tagRFP PAB-1 in the pharyngeal muscles.In young animals, tagRFP PAB-1 was mainly diffusely located throughout the pharynx. With age, a progressive accumulation in bright puncta starting in the posterior pharyngeal bulb. During aging, we also observed aggregation in the anterior pharyngeal bulb in an increasing number of animals.Quantification of different tagRFP PAB-1 aggregation levels with age in wild type and daf-2 mutant background reveals strongly delayed tagRFP PAB-1 aggregation with age in long lived animals. After watching this video, you should have a good understanding of how to collect a large number of worms for protein extraction and how to isolate highly insoluble large aggregates for further analysis by mass spectrometry or SDS page.

Research

JoVE Journal

Biology

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

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 
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Basic Caenorhabditis elegans Methods: Synchronization and Observation

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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4D Imaging of Protein Aggregation in Live Cells

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Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

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

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In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

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Methods to Assess Subcellular Compartments of Muscle in C. elegans

Methods to Assess Subcellular Compartments of Muscle in C. elegans

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Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

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 ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

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 ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

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