Name: Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans (Video) | JoVE Journal
Uploaded: 2017-11-26T13:00:00.000+00:00
Duration: 11 min 57 s
Description: Learn to detect changes in protein insolubility with age in Caenorhabditis elegans using targeted gene knockdown techniques. This method enables researchers to study proteostasis and aging mechanisms.

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5 minutes

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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</ol>

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 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.

<table><tbody><tr><td>Fernbach culture flask&amp;nbsp;</td><td>Corning</td><td>4425-2XL</td><td>Pyrex, Capacity 2,800 ml, with 3 baffle indents</td></tr><tr><td>Membrane Screw Cap&amp;nbsp;</td><td>Schott</td><td>1088655</td><td>GL45</td></tr><tr><td>Nutating Mixer</td><td>VWR</td><td>444-0148</td><td></td></tr><tr><td>Separatory funnel</td><td>Nalgene</td><td>4300-1000</td><td>Capacity 1,000 ml</td></tr><tr><td>1 ml syringe&amp;nbsp;</td><td>BD Plastipak</td><td>300013</td><td></td></tr><tr><td>Gray needle, 27 G x &amp;frac12; &quot;, 0.4 mm x 13 mm</td><td>BD Microlance 3</td><td>300635</td><td></td></tr><tr><td>Membrane filters 0.025 &amp;micro;M</td><td>Millipore</td><td>VSWP04700</td><td></td></tr><tr><td>pH strip</td><td>Machery-Nagel</td><td>92110</td><td>pH-Fix 0-14</td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Roche</td><td>4693132001</td><td>Complete Mini EDTA-free tablets&amp;nbsp;</td></tr><tr><td>Octoxynol-9&amp;nbsp;</td><td>Applichem</td><td>A1388</td><td>Triton X-100</td></tr><tr><td>4-Morpholineethanesulfonic acid (MES)</td><td>Sigma-Aldrich</td><td>M1317</td><td></td></tr><tr><td>Nonylphenylpolyethylenglycol</td><td>Applichem</td><td>A1694</td><td>Nonidet P40 (NP40)</td></tr><tr><td>DNaseI</td><td>Roche</td><td>04716728001</td><td>recombinant, RNase free</td></tr><tr><td>RNaseA</td><td>Promega</td><td>A7973</td><td>solution</td></tr><tr><td>Total protein blot staining</td><td>Thermofisher</td><td>S11791</td><td>Sypro Ruby protein blot stain</td></tr><tr><td>Total protein gel staining</td><td>Thermofisher</td><td>S12001</td><td>Sypro Ruby protein gel stain</td></tr><tr><td>TCEP (tris (2-carboxyethyl) phosphine hydrochloride)</td><td>Serva</td><td>36970</td><td></td></tr><tr><td>Iodoacetamide</td><td>Serva</td><td>26710</td><td></td></tr><tr><td>Ammoniumbicarbonate</td><td>Sigma-Aldrich</td><td>09830</td><td></td></tr><tr><td>Sequencing Grade Modified Trypsin</td><td>Promega</td><td>V5111</td><td></td></tr><tr><td>Isobaric tags for relative and absolute quantitation</td><td>Sciex</td><td>4352135</td><td>iTRAQ Reagents Multiplex Kit</td></tr><tr><td>Centrifuge Avanti J-26XP</td><td>Beckmann Coulter</td><td>393126</td><td></td></tr><tr><td>Ultracentrifuge Optima Max-XP</td><td>Beckmann Coulter</td><td>393315</td><td></td></tr><tr><td>Centrifuge 5424R</td><td>Eppendorf</td><td>5404000413</td><td></td></tr><tr><td>Centrifuge 5702</td><td>Eppendorf</td><td>5702000329</td><td></td></tr><tr><td>Centrifuge Megafuge 40R</td><td>Thermo Scientific</td><td>75004518</td><td></td></tr><tr><td>Concentrator Plus</td><td>Eppendorf</td><td>5305000304</td><td>Centrifugal evaporator</td></tr><tr><td>Fluorescent stereo-microscope M165 FC&amp;nbsp;</td><td>Leica</td><td></td><td>With Planapo 2.0x objective</td></tr><tr><td>Dissection microscope</td><td>Leica&amp;nbsp;</td><td></td><td>Leica S6E</td></tr><tr><td>High magnification microscope Zeiss Axio Observer Z1</td><td>Zeiss</td><td></td><td>With PlanAPOCHROMAT 20x objective and Zeiss Axio Cam MRm</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Image analysis software</td><td>ImageJ</td><td></td><td></td></tr><tr><td>Analysis of mass spectrometry data</td><td>Protein Prospector</td><td></td><td>http://prospector.ucsf.edu/prospector/mshome.htm</td></tr><tr><td>&lt;strong&gt;E.coli strain&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>OP50</td><td>CGC</td><td></td><td></td></tr><tr><td>&lt;strong&gt;RNAi bacteria&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>L4440</td><td>Julie Ahringer RNAi library</td><td></td><td></td></tr><tr><td>&lt;strong&gt;&lt;em&gt;C. elegans&lt;/em&gt; mutants&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CF2253</td><td>CGC, strain name: EJ1158&amp;nbsp;</td><td></td><td>Genotype: &lt;em&gt;gon-2(q388)&lt;/em&gt;</td></tr><tr><td>&lt;strong&gt;&lt;em&gt;C. elegans&lt;/em&gt; transgenics&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DCD214</td><td>Della David&#039;s lab at DZNE T&amp;uuml;bingen</td><td></td><td>Genotype: &lt;em&gt;N2; uqIs24[Pmyo-2::tagrfp::pab-1]&lt;/em&gt;</td></tr><tr><td>DCD215</td><td>Della David&#039;s lab at DZNE T&amp;uuml;bingen</td><td></td><td>Genotype: &lt;em&gt;daf-2(e1370) III; uqIs24[Pmyo-2::tagrfp::pab-1]&lt;/em&gt;</td></tr></tbody></table>

methods to study changes in inherent protein aggregation with age in caenorhabditis elegans

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...

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

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

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Biology

This video demonstrates an in vivo imaging-based assay to determine proteostasis decline due to aging. The assay uses Caenorhabditis elegans expressing polyglutamine YFP-fusion protein in body wall muscles to measure the age-associated aggregation of polyglutamine and induction of proteotoxicity, leading to paralysis.

Fluorescent Reporter-Based Paralysis Assay: A Technique to Assess Age-Associated Progressive Formation of Polyglutamine Fluorescent Reporter and Associated Paralysis in Caenorhabditis elegans

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.

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

Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease models.

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

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

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

Automating Aggregate Quantification in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

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

We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

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

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

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

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Learn to detect changes in protein insolubility with age in Caenorhabditis elegans using targeted gene knockdown techniques. This method enables researchers to study proteostasis and aging mechanisms.

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

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