Name: investigating the spreading and toxicity of prion-like proteins using the metazoan model organism c. elegans
Uploaded: 2015-01-08T14:00:00.000+00:00
Duration: 12 min 57 s
Description: Watch this Scientific Journal Video about Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans at JoVE.com

We thank Cindy Voisine and Yoko Shibata for helpful discussion and critical comments on the manuscript. We acknowledge the High Throughput Analysis Laboratory (HTAL) and the Biological Imaging Facility (BIF) at Northwestern University for their assistance. This work was funded by grants from the National Institutes of Health (NIGMS, NIA, NINDS), the Ellison Medical Foundation, and the Daniel F. and Ada L. Rice Foundation (to R.I.M.). C.I.N.-K. was supported by the Deutsche Forschungsgemeinschaft (KR 3726/1-1).

The authors declare no competing financial interests.

الأساليب المذكورة هنا تساعد على توضيح نشر والخلية معقدة المستقلة وغير المستقلة سمية الخلايا من البروتينات مثل بريون. اكتشفنا مؤخرا أن يؤخذ على عصاري خلوي مجال بريون المعرضة للتجميع ما يصل إلى الحويصلات بغشاء في عملية الالتهام الذاتي ذات الصلة. مجموعة فرعية معينة من...

وترتبط العديد من الأمراض العصبية، بما في ذلك مرض الزهايمر (AD)، ومرض باركنسون (PD)، الضموري العضلي التصلب الجانبي (ALS)، والاعتلال الإسفنجي المعدية (TSEs)، مع البروتينات المعرضة للتجميع، وبالتالي تعرف باسم البروتين misfolding اضطرابات (تطوير إدارة الأداء ). TSEs أو أمراض بريون تشكل فئة فريدة من تطوير إدارة الأداء في أنها يمكن أن تكون معدية في كل من البشر والحيوانات 1. على المستوى الجزيئي، والبريونات تكرار من خلال تجنيد وتحويل أحادى α-الحلزون الغنية المشفرة المضيف بي ار بي الخلوي (بي ار بي C) في المرضية ورقة β الغنية بي ار بي الشوري التشكل 2،3. وقد تم تحديد المجاميع البروتين نشر النفس أيضا في الفطريات، التي تشترك في الخصائص المهمة مع البريونات الثدييات 4،5. بالإضافة إلى ذلك، والبريونات الثدييات قادرة على الانتقال من خلية الى خلية وتصيب خلايا ساذجة 6،7. في حين تطوير إدارة الأداء الغيرإيه من TSEs ليست معدية، إلا أنهما يشتركان مبدأ المسببة للأمراض مشتركة مع الأمراض بريون 8،9. على الرغم من أن البروتينات ترتبط كل من تطوير إدارة الأداء لا ترتبط في هيكل أو وظيفة، وأنهم جميعا المجاميع شكل من خلال عملية تشبه بلورة تسمى منوى أو المصنفة البلمرة. وعلاوة على ذلك البذور البروتينية تنمو من خلال تجنيد الإسوية القابلة للذوبان على 2،10،11. كفاءة لنشر الذاتي تختلف في الجسم الحي، وهذا يتوقف على الخصائص الجوهرية للبروتين، والتي جنبا إلى جنب مع العوامل الخلوية إضافية مثل المحرمين الجزيئي تحدد في نهاية المطاف معدلات التنوي الكلي، البذر، والتجزؤ ونشر 12-15. وبالتالي، يجب ان يكون هناك توازن دقيق بين هذه العوامل التي تسمح نشر كفاءة من البروتين التجميع. قد يفسر هذا أيضا لماذا فقط بعض المجاميع amyloidogenic تؤوي خصائص بريون، وبالتالي ليس كل تطوير إدارة الأداء والمعدية. ويبدو أن البريونات لتمثيل س &#39;أعلى فناني الأداءاتحاد كرة القدم طيف واسع من المجاميع البروتينية الذاتي تكرار، مما يجعلها أداة قوية لدراسة تطوير إدارة الأداء 8،13. يثير الاهتمام والفضول، وسمية المرتبطة المجاميع المرتبطة المرض غالبا ما يكون غير خلية عنصر مستقل 16،17. وهذا يعني أنها تؤثر على الخلايا المجاورة التي لا تعبر عن الجين المقابل، على النقيض من تأثير مستقل الخلية بدقة، مما يعني أن فقط الخلايا معربا عن المعرض الجين النمط الظاهري محدد. وقد تجلى ذلك بشكل مقنع عن طريق التعبير الأنسجة محددة أو هدم البروتينات منها في نماذج عديدة من أمراض الاعصاب 18-26. وقد اقترحت آليات مختلفة كأساس لهذه الخلايا غير سمية الحكم الذاتي في تطوير إدارة الأداء، بما في ذلك إمدادات تقلص المواد الغذائية، وخلل في الإشارات العصبية، الغلوتامات التحفيز الزائدة، وneuroinflammation 16،27،28. وبالإضافة إلى ذلك، migh حركة تشبه بريون من المجاميع المرتبطة المرض بين الخلايار المساهمة في هذا الجانب 29،30. وتشير أدلة متزايدة على أن الادراج البروتين أخرى من البريونات يمكن أن تنقل من خلية الى خلية، وهو ما قد يفسر نشر سمة من الأمراض التي لوحظت في العديد تطوير إدارة الأداء 30-36. ومع ذلك، لم يتم بعد تحديد ما إذا كانت هناك علاقة سببية واضحة بين الحركة بين الخلايا البروتينات المرض وتأثير سام على الخلايا المجاورة. ولذلك، فهم أفضل للمسارات الخلوية التي تكمن وراء انتقال خلية الى خلية خلية وغير سمية الحكم الذاتي أمر ضروري وأساسي لتطوير علاجات جديدة. ومع ذلك، لا يفهم كثير من جوانب بريون مثل نشر والخلوية العوامل التي تؤثر على انتقال الخلية الى خلية من البروتينات تتجمع في metazoans جيدا، ولا سيما على مستوى العضوي. الديدان الخيطية ايليجانس انواع معينة لديها العديد من المزايا التي توفر إمكانات ل اكتشاف جوانب جديدة من تشبه بريون SPREADIنانوغرام في metazoans 17. انها شفافة، والسماح لفي الجسم الحي تتبع البروتينات من الموسومة fluorescently في الكائن الحي. وعلاوة على ذلك، يتم الحفاظ العديد من العمليات الخلوية والفسيولوجية المتضررين من المرض من الديدان إلى الإنسان، وC. ايليجانس هي أيضا قابلة للطائفة واسعة من التلاعب الجيني والتحليلات الجزيئية والكيمياء الحيوية 37-39. بالضبط 959 الخلايا الجسدية تشكل خنثى الكبار مع خطة الهيئة البسيطة التي لا يزال لديها العديد من أنواع الأنسجة متميزة، بما في ذلك العضلات، والخلايا العصبية والأمعاء. لوضع نموذج جديد في بريون C. ايليجانس، اخترنا للتعبير عن خارجيا وتتميز كذلك الجلوتامين / الأسباراجين (Q / N) الغنية بريون NM المجال من عصاري خلوي بروتين بريون Sup35 الخميرة، حيث لا توجد بروتينات بريون الذاتية المعروفة في الديدان 4،40. وقد البريونات الخميرة لا تقدر بثمن في توضيح الآليات الأساسية لتكرار بريون 41-44. وعلاوة على ذلك، NM هو التنوبالحادي وعصاري خلوي البروتين مثل بريون التي قد تظهر لتلخيص دورة الحياة الكاملة للبريون في الثقافة خلايا الثدييات 45،46. وبالمثل، عندما أعرب في C. ايليجانس، المجال بريون Sup35 اعتمدت بشكل جيد لمتطلبات مختلفة للنشر في الخلايا metazoan بالمقارنة مع خلايا الخميرة والملامح الرئيسية عرضت من بريون البيولوجيا ارتبط 40. NM التجميع مع النمط الظاهري السامة العميق، بما في ذلك تعطيل سلامة الميتوكوندريا ومظهر من مختلف الحويصلات الالتهام الذاتي ذات الصلة على المستوى الخلوي، فضلا عن اعتقال الجنينية واليرقات، تأخر في النمو، واضطراب واسع النطاق للبيئة طي البروتين على المستوى العضوي. لافت للنظر، يسلك المجال بريون خلية مستقلة وغير خلية سمية مستقلة، مما يؤثر على الأنسجة المجاورة التي لم أعرب التحوير. وعلاوة على ذلك، يتم مراقبة النقل حويصلي في مجال بريون داخل وبين خلايا في الوقت الحقيقي &lt;em&gt; في الجسم الحي 40. نحن هنا وصف كيفية فحص نشر مثل بريون في C. ايليجانس. سوف نشرح كيفية مراقبة النقل داخل الأقاليم وفيما بين الخلايا من الحويصلات التي تحتوي على المجال بريون باستخدام الوقت الفاصل بين مضان المجهري. سوف نؤكد على أن استخدام أجهزة الاستشعار الأنسجة محددة قابلة للطي وأعرب بتواجد مطلق للصحفيين الإجهاد لتقييم آثار خلية خلية مستقلة تتمتع بالحكم الذاتي وغير على اللياقة البدنية الخلوية. وأخيرا، فإننا سوف تصف الإجراء من الجينوم واسعة تدخل الحمض النووي الريبي (رني) شاشة أجريت مؤخرا لتحديد معدلات جديدة من سمية التي يسببها بريون. في الجمع، ويمكن لهذه الأساليب تساعد على ندف بصرف النظر مسارات الجينية التي تساهم في الحركة بين الخلايا البروتينات وغير خلية سميتها مستقلة.

<table><tbody><tr><td>Reagent</td><td></td><td></td><td></td></tr><tr><td>Nanosphere size standards 100 nm</td><td>ThermoScientific</td><td>3100A</td><td></td></tr><tr><td>Levamisole</td><td>Sigma</td><td>L-9756</td><td></td></tr><tr><td>IPTG</td><td>Sigma</td><td>15502-10G</td><td></td></tr><tr><td>Ahringer RNAi library</td><td>Source BioScience LifeSciences&amp;nbsp;</td><td></td><td>http://www.lifesciences.sourcebioscience&lt;br/&gt; .com/clone-products/non-mammalian/c-elegans/c-elegans-rnai-library/</td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Sorvall Legend XTR Refrigerated Centrifuge, 120VAC</td><td>ThermoScientific</td><td>75004521</td><td>http://www.coleparmer.com/Product/Thermo_Scientific_Sorvall_Legend_&lt;br/&gt; XTR_Refrigerated_Centrifuge_120&lt;br/&gt; VAC/EW-17707-60</td></tr><tr><td>96 pin replicator&amp;nbsp;</td><td>Scionomix&amp;nbsp;&amp;nbsp;</td><td></td><td>http://www.scinomix.com/all-products/96-pin-replicator/</td></tr><tr><td>HiGro high-capacity, incubating shaker&amp;nbsp;</td><td>Digilab</td><td></td><td>http://www.digilabglobal.com/higro</td></tr><tr><td>Multidrop Combi Reagent Dispenser&amp;nbsp;</td><td>Titertrek</td><td></td><td>http://groups.molbiosci.northwestern.edu/hta/titertek.htm</td></tr><tr><td>Biomek FX AP96 Automated Workstation&amp;nbsp;</td><td>Beckman Coulter</td><td></td><td>http://groups.molbiosci.northwestern.edu/hta/biomek_multi.htm</td></tr><tr><td>Innova44 shaker</td><td>New Brunswick</td><td></td><td>http://www.eppendorf.com/int///index.php?sitemap=2.3&amp;amp;pb=d78efbc05310ec&lt;br/&gt; 04&amp;amp;action=products&amp;amp;contentid=1&amp;amp;&lt;br/&gt; catalognode=83389</td></tr><tr><td>M205 FA&amp;nbsp;</td><td>Leica</td><td></td><td>http://www.leica-microsystems.com/de/produkte/stereomikroskope-makroskope/fluoreszenz/details/product/leica-m205-fa/</td></tr><tr><td>ORCA-R2 C10600-10BDigital CCD camera</td><td>Hamamatsu</td><td></td><td>http://www.hamamatsu.com/jp/en/community/life_science_camera/product/search/C10600-10B/index.html</td></tr><tr><td>Spinning Disc AF Confocal Microscope&amp;nbsp;</td><td>Leica</td><td></td><td>http://www.leica-microsystems.com/products/light-microscopes/life-science-research/fluorescence-microscopes/details/product/leica-sd-af/</td></tr><tr><td>Falcon 4M60 camera&amp;nbsp;</td><td>Teledyne Dalsa&amp;nbsp;</td><td></td><td>http://www.teledynedalsa.com/imaging/products/cameras/area-scan/falcon/PT-41-04M60/</td></tr><tr><td>Software</td><td></td><td></td><td></td></tr><tr><td>MetaMorph Microscopy Automation &amp;amp; Image Analysis Software</td><td>Molecular Devices</td><td></td><td>http://www.moleculardevices.com/products/software/meta-imaging-series/metamorph.html</td></tr><tr><td>Hamamatsu SimplePCI Image Analysis Software</td><td>Meyer Instruments</td><td></td><td>http://meyerinst.com/imaging-software/hamamatsu/index.htm</td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td>http://rsbweb.nih.gov/ij/download.html</td></tr><tr><td>wrMTrck plugin for ImageJ</td><td></td><td></td><td>http://www.phage.dk/plugins/wrmtrck.html</td></tr><tr><td>&lt;em&gt;C. elegans&lt;/em&gt; strains</td><td></td><td></td><td></td></tr><tr><td>N2 (WT)</td><td>Caenorhabditis Genetics Center (CGC)</td><td></td><td>http://www.cgc.cbs.umn.edu/strain.php?id=10570</td></tr><tr><td>AM815&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; rmIs323[myo-3p::sup35(r2e2)::rfp]</td><td>Morimoto lab</td><td></td><td>available from our laboratory&amp;nbsp;</td></tr><tr><td>See table 1 for a source for folding sensor and stress reporter strains</td><td></td><td></td><td></td></tr></tbody></table>

investigating the spreading and toxicity of prion-like proteins using the metazoan model organism c. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

مراقبة بيخلوي نشر من البروتينات مثل بريون من قبل في الجسم الحي الوقت الفاصل بين التصوير المعدلة وراثيا C. خطوط ايليجانس معربا عن المجال بريون بشكل خاص مناسبة تماما لتحليل جوانب معينة من مثل بريون البروتين?...

Watch this Scientific Journal Video about Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans at JoVE.com

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

التحقيق في نشر وسمية البروتينات مثل بريون باستخدام Metazoan نموذج حي<em&gt; C. ايليجانس</em

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

investigating-spreading-toxicity-prion-like-proteins-using-metazoan

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

15.8K Views.  Northwestern University. Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

Video: Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

يعاير β اميلويد السمية باستخدام المعدلة وراثيا C. ايليجانس النموذجي

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

Neuroscience

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

A<em&gt; انواع معينة ايليجانس</em&gt; النظام النموذجي للدراسات Amylopathy

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological conditions. Post-mitotic neurons represent a major cell type affected by aging. Although multiple mammalian models of neuronal aging exist, they are challenging and expensive to establish. The roundworm Caenorhabditis elegans is a powerful model to study neuronal aging, as these animals have short lifespan, an available robust genetic toolbox, and well-cataloged nervous system. The method presented herein allows for seamless isolation of specific cells based on the expression of a transgenic green fluorescent protein (GFP). Transgenic animal lines expressing GFP under distinct, cell type-specific promoters are digested to remove the outer cuticle and gently mechanically disrupted to produce slurry containing various cell types. The cells of interest are then separated from non-target cells through fluorescence-activated cell sorting, or by anti-GFP-coupled magnetic beads. The isolated cells can then be cultured for a limited time or immediately used for cell-specific ex vivo analyses such as transcriptional analysis by real time quantitative PCR. Thus, this protocol allows for rapid and robust analysis of cell-specific responses within different neuronal populations in C. elegans.

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

Here, we present a protocol for simple isolation of specific groups of live neuronal cells expressing green fluorescent protein from transgenic Caenorhabditis elegans lines. This method enables a variety of ex vivo studies focused on specific neurons and has the capacity to isolate cells for further short-term culturing.

عزل مجموعات الخلايا العصبية المحددة من الدودة المستديرة Caenorhabditis elegans

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological ...

Developmental Biology

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.

مراقبة حركية تجميع البروتين في الجسم الحي باستخدام عد التضمين الآلي في 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 ...

Biochemistry

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. Here, a Drosophila melanogaster model that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach we describe here can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.

رصد خلية إلى انتقال بروتين بريون مثل المجاميع في Melanogaster المورفولوجية

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

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 ...

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent&#160;model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a&#160;knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::G&#945;S(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

In a C. elegans excitotoxicity model, this protocol employs in vivo imaging to analyze the regulation of necrotic neurodegeneration, the effect of genes encoding candidate mediators, and involvement of mitochondria. Cell dissociation and sorting is used to specifically obtain at-risk neurons for cell-specific transcriptomic analysis of neurodegeneration and neuroprotection mechanisms.

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of 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.

Caenorhabditis elegans كنظام نموذجي لاكتشاف المركبات النشطة بيولوجيا ضد السمية العصبية بوساطة البولي جلوتامين

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

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.

النمذجة العمر المرتبطة الأمراض العصبية في Caenorhabditis elegans

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

An Introduction to Caenorhabditis elegans

Caenorhabditis elegans is a microscopic, soil-dwelling roundworm that has been powerfully used as a model organism since the early 1970&rsquo;s. It was initially proposed as a model for developmental biology because of its invariant body plan, ease of genetic manipulation and low cost of maintenance. Since then C. elegans has rapidly grown in popularity and is now utilized in numerous research endeavors, from studying the forces at work during locomotion to studies of neural circuitry.
This video provides an overview of basic C. elegans biology, a timeline of the many milestones in its short but storied history, and finally a few exciting applications using C. elegans as a model organism.

Caenorhabditis elegans Round Worm as a Model Organism

Caenorhabditis elegans is a microscopic, soil-dwelling roundworm that has been powerfully used as a model organism since the early 1970&rsquo;s.  It was ...

التحقيق في نشر وسمية البروتينات مثل بريون باستخدام Metazoan نموذج حي

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يعاير β اميلويد السمية باستخدام المعدلة وراثيا C. ايليجانس النموذجي

A

عزل مجموعات الخلايا العصبية المحددة من الدودة المستديرة Caenorhabditis elegans

مراقبة حركية تجميع البروتين في الجسم الحي باستخدام عد التضمين الآلي في Caenorhabditis elegans

رصد خلية إلى انتقال بروتين بريون مثل المجاميع في Melanogaster المورفولوجية

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

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Caenorhabditis elegans كنظام نموذجي لاكتشاف المركبات النشطة بيولوجيا ضد السمية العصبية بوساطة البولي جلوتامين

النمذجة العمر المرتبطة الأمراض العصبية في Caenorhabditis elegans

An Introduction to Caenorhabditis elegans