Bu eser DZNE ve Marie Curie International Reintegration Grant (D.C.D. 322120) fon tarafından desteklenmiştir

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorodeoxyuridine+enhances+the+heat+shock+response+and+decreases+polyglutamine+aggregation+in+an+HSF-1-dependent+manner+in+Caenorhabditis+elegans.">Fluorodeoxyuridine enhances the heat shock response and decreases polyglutamine aggregation in an HSF-1-dependent manner in Caenorhabditis elegans.</a> Mech Ageing Dev. 141, 1-4 (2014).</li><li>Angeli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+DNA+synthesis+inhibitor+is+protective+against+proteotoxic+stressors+via+modulation+of+fertility+pathways+in+Caenorhabditis+elegans.">A DNA synthesis inhibitor is protective against proteotoxic stressors via modulation of fertility pathways in Caenorhabditis elegans.</a> Aging (Albany NY). 5, 759-769 (2013).</li><li>Feldman, N., Kosolapov, L., Ben-Zvi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorodeoxyuridine+improves+Caenorhabditis+elegans+proteostasis+independent+of+reproduction+onset.">Fluorodeoxyuridine improves Caenorhabditis elegans proteostasis independent of reproduction onset.</a> PLoS One. 9, 85964 (2014).</li><li>Davies, S. K., Leroi, A. M., Bundy, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorodeoxyuridine+affects+the+identification+of+metabolic+responses+to+daf-2+status+in+Caenorhabditis+elegans.">Fluorodeoxyuridine affects the identification of metabolic responses to daf-2 status in Caenorhabditis elegans.</a> Mech Ageing Dev. 133, 46-49 (2012).</li><li>Luo, S., Kleemann, G. A., Ashraf, J. M., Shaw, W. M., Murphy, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+and+insulin+signaling+regulate+reproductive+aging+via+oocyte+and+germline+quality+maintenance.">TGF-beta and insulin signaling regulate reproductive aging via oocyte and germline quality maintenance.</a> Cell. 143, 299-312 (2010).</li><li>Andux, S., Ellis, R. 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Yazarlar ifşa gerek yok.

Burada son derece çözünmez protein toplamları RNAi için analiz için kütle spektrometresi ve Western Blot tarafından tabi C. elegans yaşlanma dan izole etmek için bir metodoloji raporu. Biz proteostasis IIS büyük ölçüde azaltarak iyileştirilmesi yaş bağımlı protein toplama engellediğini gösterir. C. elegansiçinde overexpress için belirli toplama eğilimli proteinler seçerek, daha fazla doğal protein toplama oransal mekanizmaları incelemek mümkündür.
<p class="jove_conten...

Protein misfolding ve toplama bir hallmark reklam, PD, amyotrofik lateral skleroz (ALS), da demans (FTD) ve diğerleri gibi çeşitli nörodejeneratif hastalıkların kabul edilmektedir. Örneğin, α-synuclein derlemeler amiloid liflerinde içine bu PD hastalarında ALS hastalar TDP-43 veya FUS misfold iken motor nöronların oluşan bozucu formu sitoplazmik toplamları için özellikle de gözlemliyorum nigra Lewy organları olarak birikir. Her bu nörodejeneratif hastalıklar, protein homeostazı veya proteostasis Bakımı mekanizmaları hastalık için sonuç olarak lider misfolded proteinlerin birikimi önlemek başarısız.
Normal şartlar altında düzenleyici mekanizmaların sıkı bir şekilde denetlemesi protein sentezi, katlama ve bozulma oranını ve Proteostasis hücresel işlev görmesini sağlamak için önemlidir. Çeşitli çalışmalarda ile yaşlanma, protein homeostazı korumak yeteneği birçok hücre ve organların yavaş yavaş açığa ve yaş proteostasis ağlarıyla fizyolojik bozulma için önemli bir ağırlaştırıcı faktör olduğunu göstermek nörodejeneratif hastalıklar (başvurular1,2,3' te gözden). Protein kalite kontrol ve gelişeceğini protein stres hücresel yanıt yaşla birlikte tehlikeye protein misfolding ve toplama yaşlanma genel bir sonucu olabilir gösteriyor. Nitekim, biz ve diğerleri protein toplama hastalığı için sınırlı değildir ve bunun yerine Proteom parçası çok deterjan-yaşlı hayvanlar4,5,6',7 çözünmez olur göstermiştir ,8,9,10. Hesaplamalı ve in vivo analizi bu fizyolojik yaşı ile ilgili toplamları çeşitli yönleri5hastalık toplamları benzer saptandı. Endojen, yaş-bağımlı protein toplama keşfi bize protein toplama, ectopically ifade insan hastalık ilişkili proteinler kullanmadan düzenleyen moleküler ve hücresel mekanizmaları incelemek için fırsat verir. Şu anda, bu bozukluk organizmanın sağlık üzerine etkisi ve yaygın protein insolubility düzenlenmesi hakkında sadece sınırlı bilgi bulunmaktadır.
Nematot C. elegans bu hayvanlar nispeten kısa bir ömrü var ve daha yüksek organizmalarda gözlenen birçok karakteristik yaşlanma özellikleri göstermek gibi araştırma yaşlanma içinde en yoğun olarak çalışılan model organizmalar biridir. Protein insolubility yaşlanma etkilerini C. elegans içinde ateş araştırma11 alan hastalık toplamları ayıklamak için yaygın olarak kullanılan farklı çözünürlük göre sıralı biyokimyasal ayırma tarafından incelenmiştir . Nicel kütle spektrometresi tarafından birkaç yüz proteinler toplama C. elegans eğilimli hastalık5yokluğunda olmak için gösterildi. Burada biz ayrıntılı olarak tarif sıvı kültür ve miktar için toplanan proteinler kütle spektrometresi ve analiz tarafından Western blot tarafından izole etmek için sıralı ayıklama solucanlar çok sayıda büyümeye protokolü. Çünkü misfolded ve toplama eğilimli proteinler yaşlı içinde birikir C. elegans gonads ve maskeleri değişikliklerinin diğer somatik dokular5,12,13, erbezi daha az mutant protein analizi odaklanmak için kullanmak Sigara üreme dokularda insolubility. Yöntemi sunulan % 0.5 SDS çözünmez ve nispeten düşük santrifüj hızlı tarafından pelleted son derece çözünmez, büyük toplamları çözümleme sağlar. Alternatif olarak, aynı zamanda daha küçük ve daha fazla çözünür toplamları toplamak için daha az sıkı bir ayıklama Protokolü oldu başka bir yerde10yayınlandı. Ayrıca, C. eleganstoplama vivo içinde değerlendirmek için kullanılan yöntem açıklamaktadır.
Genel olarak, bu yöntemleri RNA müdahale (RNAi) ile birlikte bir gen yaş bağımlı protein toplama oransal ilgi rolü değerlendirebilir. Bunun için biz genç ve yaşlı kurt ve nakavt RNAi kullanarak ilgi belirli bir protein olmayan özler analizini açıklar. Bu yöntemler proteostasis ağ bileşenlerini protein insolubility düzenleyen belirlemek için güçlü bir araç olmalıdır. Birkaç müdahaleler gibi insülin/İnsülin benzeri büyüme faktörü (IGF) azaltılmış 1 (IIS) sinyal gösterilmiştir dramatik C. elegans yaşlanma14geciktirmek için. Uzun ömürlü yolları genellikle protein-kalite kontrol mekanizmaları teşvik ve böylece bu yolları aktif protein toplama oranını etkileyen. Örneğin, sınırlı doğal protein toplama IIS yolu7inhibisyonu üzerine uzun ömürlü hayvanlarda göstermektedir.

<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

Son yıllarda, nörodejeneratif hastalıkları, Alzheimer hastalığı (Ah) ve Parkinson hastalığı (PD), gibi yaygınlığı büyüdü. Bu yaş ilişkili bozuklukları protein toplamları bu hastaların beynindeki fibrillary yapısı ile görünümünü ile karakterizedir. Tam olarak normal çözünür proteinler tabi neden bir toplama işlemi kötü anlaşılır kalır. Protein toplama hastalık süreçlerine sınırlı değildir ve bunun yerine normal yaşlanma sürecinin bir parçası protein toplama, ectopically kullanmadan düzenleyen moleküler ve hücresel mekanizmaları çalışma sağlar keşif insan ifade hastalık ilişkili proteinler. Burada Caenorhabditis elegans doğal protein toplamada tamamlayıcı yaklaşımlar ile incelemek için yöntemleri açıklanmaktadır. İlk olarak, biz yaşlı hayvanlar elde etmek için çok sayıda yaş senkronize C. elegans yetiştirmeyi incelemek ve biz son derece-çözünmez-büyük toplamları ayırmak için biyokimyasal yordamlar mevcut. Hedeflenen genetik nakavt ile birlikte, teşvik ya da yaş bağımlı protein toplama nicel kütle spektrometresi ile ya da kapsamlı bir çözümlemesini kullanarak engelleyen bir gen rolünü incelemek mümkündür veya bir aday tabanlı analiz antikorlar ile. Bu bulgular sonra toplama eğilimli proteinler floresan öğesini ifade Transjenik hayvanlar ile vivo içinde analiz tarafından onaylanır. Bu yöntemler belirli proteinler topluluğu ile yaş ve sonuçta bu proteinler tamamen işlevsel tutmayı yatkındır neden açıklamak yardımcı olmalıdır.

Biz burada ne kadar uzun ömürlü hayvanlarla değerlendirmek için sunulan yöntemleri kullanılan azaltılmış IIS modüle yaş bağımlı protein toplama. Tarafından Western blot (2.2, çabuk çözünmez protein çıkarma Western blot analizi için bkz. adım), toplam ve genç (3. gün erişkin) çözünmez protein içeriği analiz ve yaş (gün 18 yetişkinlik) solucanlar kontrol RNAi ve insülin hedefleme RNAi / IGF-1-benzeri reseptör daf-2. RNAi koşulları (<strong cla...

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

Yöntemleri ile Caenorhabditis elegans çağda doğal Protein toplama çalışma değişikliklere

Burada sunulan yöntem model organizma C. elegansnormal yaşlanma sırasında protein toplama keşfetmek için hedeftir. Protokolü temsil eden yaş ile oluşturmak son derece çözünmez büyük toplamları çalışmaya ve nasıl protein toplama proteostasis değişiklikleri etkisini belirlemek için güçlü bir araç.

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

methods-to-study-changes-inherent-protein-aggregation-with-age

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

8.4K Görüntüleme.  German Center for Neurodegenerative Diseases (DZNE). Burada sunulan yöntem model organizma C. elegansnormal yaşlanma sırasında protein toplama keşfetmek için hedeftir. Protokolü temsil eden yaş ile oluşturmak son derece çözünmez büyük toplamları çalışmaya ve nasıl protein toplama proteostasis değişiklikleri etkisini belirlemek için güçlü bir araç.

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

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

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. We present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells, such as neurons, in a noninvasive manner and with the progression of aging and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which results in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.

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

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

Yaşlanma C. Floresan Yaşam Boyu Görüntüleme Kullanarak Elegans Amiloid Yapıların Karakterizasyonu

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These ...

Biyokimya

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

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

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

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

Caenorhabditis elegans'ta Otomatik Inklüzyon Sayımı Kullanarak Protein Agregasyon Kinetiğinin In Vivo'da İzlenmesi

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis ...

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.

Temel<em&gt; Caenorhabditis elegans</em&gt; Yöntem: Senkronizasyon ve Gözlem

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

Biyoloji

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 ...

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s disease (HD) is principally driven by a CAG nucleotide repeat that encodes an expanded glutamine tract in huntingtin protein. Thus, the inhibition of polyglutamine (polyQ) aggregation and, in particular, aggregation-associated neurotoxicity is a useful strategy for the prevention of HD and other polyQ-associated conditions. This paper introduces generalized experimental protocols to assess the neuroprotective capacity of test compounds against HD using established polyQ transgenic Caenorhabditis elegans models. The AM141 strain is chosen for the polyQ aggregation assay as an age-associated phenotype of discrete fluorescent aggregates can be easily observed in its body wall at the adult stage due to muscle-specific expression of polyQ::YFP fusion proteins. In contrast, the HA759 model with strong expression of polyQ-expanded tracts in ASH neurons is used to examine neuronal death and chemoavoidance behavior. To comprehensively evaluate the neuroprotective capacity of target compounds, the above test results are ultimately presented as a radar chart with profiling of multiple phenotypes in a manner of direct comparison and direct viewing.

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Here, we present a protocol to assess the neuroprotective activities of test compounds in Caenorhabditis elegans, including polyglutamine aggregation, neuronal death, and chemoavoidance behavior, as well as an exemplary integration of multiple phenotypes.

Poliglutamin aracılı nörotoksisiteye karşı biyoaktif bileşiklerin keşfedilmesi için bir model sistem olarak Caenorhabditis elegans

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s ...

Nörobilim

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.

Caenorhabditis eleganlarında Dokuya Özgü Proteostatik Düşüşün Ölçülmesi

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

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 detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases 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.

Caenorhabditis elegans Yaş İlişkili Nörodejeneratif Hastalıkların Modellendirilmesi

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

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

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

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

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

Caenorhabditis elegans de novo Protein Sentez Oranlarının Değerlendirilmesi

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

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

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

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

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

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

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification 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.

Caenorhabditis elegans'ta Agrega Niceliğinin Otomatikleştirilmesi

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

Yöntemleri ile Caenorhabditis elegans çağda doğal Protein toplama çalışma değişikliklere

Özet

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Bu videodaki bölümler

Yaşlanma C. Floresan Yaşam Boyu Görüntüleme Kullanarak Elegans Amiloid Yapıların Karakterizasyonu

Caenorhabditis elegans'ta Otomatik Inklüzyon Sayımı Kullanarak Protein Agregasyon Kinetiğinin In Vivo'da İzlenmesi

Temel

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

Poliglutamin aracılı nörotoksisiteye karşı biyoaktif bileşiklerin keşfedilmesi için bir model sistem olarak Caenorhabditis elegans

Caenorhabditis eleganlarında Dokuya Özgü Proteostatik Düşüşün Ölçülmesi

Caenorhabditis elegans Yaş İlişkili Nörodejeneratif Hastalıkların Modellendirilmesi

Caenorhabditis elegans de novo Protein Sentez Oranlarının Değerlendirilmesi

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

Caenorhabditis elegans'ta Agrega Niceliğinin Otomatikleştirilmesi