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.

השיטות שתוארו כאן לעזור כדי להמחיש מתפשטים ורעילות אוטונומית תא התא המורכב אוטונומית ולא של חלבונים כמו פריון. לאחרונה גילינו שתחום פריון cytosolic צבירה מועדת הוא נלקח לתוך שלפוחית ​​קרום הנכנס בתהליך autophagy קשור. תת-קבוצה מסוימת של שלפוחית ​​אלה משלוחי תחום הפריון בת...

מחלות רבות ניווניות, כוללים מחלת אלצהיימר, מחלת פרקינסון (PD) (AD), טרשת לרוחב amyotrophic (ALS), וencephalopathies transmissible spongiform (TSEs), המשויכות חלבוני צבירה מועדת ומכאן ידועות קולקטיבי חלבון misfolding הפרעות (PMDs ). TSEs או מחלות פריון מהוות מעמד ייחודי של PMDs בכך שהם יכולים להיות זיהומיות, באדם ובעלי החיים 1. ברמה המולקולרית, פריונים לשכפל על ידי גיוס והמרת PRP monomeric α-helix-עשיר בקידוד המארח הסלולרי (PRP C) לקונפורמציה PRP Sc β גיליון העשיר פתולוגי 2,3. אגרגטים חלבון עצמי הפצת גם זוהו בפטריות, אשר חולקות מאפיינים חשובים עם פריונים יונקים 4,5. בנוסף, פריונים יונקים מסוגלים לנוע מתא אל תא ולהדביק תאים נאיביים 6,7. בעוד oth PMDsאה מ TSEs אינו מדבק, הם חולקים עיקרון פתוגניים משותף עם מחלות פריון 8,9. למרות שהחלבונים הקשורים לכל אחד מPMDs אינם קשורים במבנה או פונקציה, הם כל אגרגטים הטופס באמצעות תהליך התגבשות כמו שנקרא גרעיניים או זרע פילמור; יתר על כן זרעים חלבוניים לגדול על ידי גיוס isoforms המסיס 2,10,11. היעילות להפיץ עצמי משתנה in vivo, בהתאם למאפיינים הפנימיים של החלבון, אשר יחד עם גורמים סלולריים נוספים כגון מלווים מולקולריים סופו של דבר יקבעו את התעריפים של התגרענות כוללת, זריעה, פיצול והפצת 12-15. מכאן, שחייב להתקיים איזון עדין בין הגורמים הללו, המאפשר התפשטות יעילה של צבירת חלבון. זה עשוי גם להסביר מדוע רק חלק אגרגטים amyloidogenic נמל המאפיינים של פריון, ולכן לא כל PMDs הם מדבקים. נראים פריונים לייצג o &#39;מלמעלה מבצעים&#39;ספקטרום רחב של fa אגרגטים חלבוניים המשכפלים את עוצמה, מה שהופך אותם כלי רב עוצמה כדי ללמוד PMDs 8,13. מעניין לגלות שההרעלה כרוכה באגרגטים הקשורים למחלות לעתים קרובות יש מרכיב אוטונומי של תאים הלא 16,17. משמעות דבר היא כי הם משפיעים על תאים שכנים, שאינו מבטאים את הגן המתאים, בניגוד להשפעה אוטונומית תא בהחלט, מה שמרמז שרק תאים המבטאים את גן התערוכה פנוטיפ הספציפי. הדבר בא לידי הביטוי משכנע על ידי ביטוי רקמות ספציפיות או להפיל של החלבונים המתאימים במספר רב של דגמים של מחלות ניווניות 18-26. מנגנונים שונים הוצעו כבסיס לרעילות זו אינה תא אוטונומית בPMDs, כוללים אספקת חומרי מזון פחת, חוסר איזון באיתות עצבית, הפעלת יתר רעילה גלוטמט, וneuroinflammation 16,27,28. בנוסף, תנועה כמו פריון-אגרגטים צמודי מחלה בין תאי might לתרום להיבט זה 29,30. ראיות מצביעות על כך שהגדלת תכלילים חלבון אחרים מאשר פריונים יכולים לשדר מתא אל תא, שעשוי להסביר את מאפיין הפצה של פתולוגיה שנצפתה בPMDs רב 30-36. עם זאת, זה עדיין לא נקבע אם יש קשר סיבתי ברור בין תנועה בין תאית של חלבוני מחלה וההשפעה הרעילה על תאי שכנים. לכן, הבנה טובה יותר של המסלולים הסלולריים העומדים בבסיס שידור ורעילות אוטונומית שאינו תא תא אל התא היא הכרחית וחיונית לפיתוח תרופות חדשניות. עם זאת, היבטים רבים של פריון כמו גורמי הפצה וסלולריים המשפיעים על העברת תא אל התא של חלבוני misfolded בmetazoans אינם מובנה היטב, בפרט ברמת האורגניזם. יש נמטודות elegans Caenorhabditis מספר יתרונות המספקים פוטנציאל ל לגלות היבטים חדשים של spreadi כמו פריון-ng בmetazoans 17. זה שקוף, המאפשר למעקב in vivo חלבונים של מתויגים fluorescently באורגניזם החי. יתר על כן, תהליכים תאיים ופיסיולוגיים רבים במחלה נשמרים מתולעים לאדם, ו- C. elegans הוא גם נוח למגוון רחב של מניפולציות גנטיות וניתוחים מולקולריים וביוכימיים 37-39. בדיוק 959 תאים סומטיים מרכיבים את אנדרוגינוס המבוגר עם תכנית גוף פשוט כי עדיין יש כמה סוגי רקמות שונים, כולל שרירים, תאי עצב ומעי. לבסס מודל פריון חדש בג elegans, בחרנו להביע אקסוגני גלוטמין / -rich פריון NM תחום של חלבון פריון שמרי cytosolic Sup35 asparagine המאופיין היטב (Q / N), שכן אין חלבוני פריון אנדוגני ידועים בתולעים 4,40. פריונים שמרים כבר לא יסולא בפז בהבהרת מנגנונים בסיסיים של שכפול פריון 41-44. יתר על כן, NM הוא האשוחחלבון st cytosolic כמו פריון-שהוכח לשחזר את מחזור החיים המלא של פריון בתרבית תאי יונקים 45,46. כמו כן, כאשר באו לידי ביטוי בג elegans, תחום פריון Sup35 אימץ להפליא לדרישות השונות לריבוי בתאים מטזואניים בהשוואה לתאי שמרים ותכונות עיקריות הציגו ביולוגיה פריון צבירת 40. NM הייתה קשורה עם פנוטיפ רעיל עמוק, כוללים השיבוש של יושרה ומראה של המיטוכונדריה שלפוחית ​​שונות הקשורים autophagy ברמה התאית, כמו גם מעצר עוברי וזחל, עיכוב התפתחותי, והפרעה נרחבת של סביבת קיפול חלבונים ברמת האורגניזם. באופן מפתיע, תחום הפריון מציג רעילות אוטונומית תא תא אוטונומי ובלתי, המשפיע על רקמות השכנות, שבי transgene לא באו לידי ביטוי. יתר על כן, תחבורת לפוחי של תחום הפריון בתוך ובין התאים היא פיקוח בזמן אמת &lt;em&gt; In vivo 40. כאן אנו מתארים כיצד לבחון הפצה כמו פריון-בג elegans. אנו נסביר כיצד לפקח על תחבורת התוך ובין תאית של שלפוחית ​​המכילה תחום הפריון באמצעות מיקרוסקופ פלואורסצנטי הזמן לשגות. אנו להדגיש את השימוש בחיישני קיפול רקמות ספציפיות וכל מקום בגוף הביעו כתבי לחץ כדי לבדוק את השפעות תא אוטונומיות ובלתי תא אוטונומיות על כושר סלולארי. לבסוף, נתאר את ההליך של מסך הגנום ביצע לאחרונה רחב התערבות RNA (RNAi) לזהות מכפילי חדשים של רעילות עקב פריון. בשילוב, שיטות אלה יכולים לעזור ללהפריד מסלולים גנטיים מעורבים בתנועה בין תאית של חלבונים והרעילות האוטונומית מסוג התאים לא.

<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.

ניטור אינטר הפצה של חלבונים כמו פריון-ידי in vivo הדמיה הזמן לשגות המהונדס C. קווי elegans להביע תחום הפריון הם גם מתאימים במיוחד לניתוח של היבטים מסוימים של חלבונים כמו פריון-, למשל, העברת תא אל התא ורעילות א?...

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.

חוקר את החלבונים כמו פריון-הפצת ורעילות של שימוש במטזואניים דגם אורגניזם<em&gt; C. elegans</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.

Assaying β עמילואיד רעילות באמצעות טראנסגנטי ג elegans דגם

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

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.

ניטור הצטברות חלבונים קינטיקה ב Vivo באמצעות ספירת הכללה אוטומטית ב Elegans Caenorhabditis

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

Biochemistry

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

אוטומטיות התנהגותית ניתוח של גדול C. elegans אוכלוסיות שימוש רחב שדה-of-view מעקב פלטפורמה

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

Immunology and Infection

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.

ניטור-לתא שידור של אגרגטים חלבון פריאון-כמו בדרוזופילה Melanogaster

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

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 ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

הקרנה תפוקה גבוהה של חלבון מבוססת ירושה cerevisiae ס

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

מדידת רעילות ב -C. אלגיה

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...

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.

כימות ירידה פרוטאוסטטית ספציפית לרקמות ב elegans Caenorhabditis

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.

דוגמנות גיל הקשורים מחלות ניווניות בתוך האלאים

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

Tissue-Specific RNAi Tools to Identify Components for Systemic Stress Signaling

Over the past decade there has been a transformative increase in knowledge surrounding the regulation of protein quality control processes, unveiling the importance of intercellular signaling processes in the regulation of cell-nonautonomous proteostasis. Recent studies are now beginning to uncover signaling components and pathways that coordinate protein quality control from one tissue to another. It is therefore important to identify mechanisms and components of the cell-nonautonomous proteostasis network (PN) and its relevance for aging, stress responses and protein misfolding diseases. In the laboratory, we use genetic knockdown by tissue-specific RNAi in combination with stress reporters and tissue-specific proteostasis sensors to study this. We describe methodologies to examine and to identify components of the cell-nonautonomous PN that can act in tissues perceiving a stress condition and in responding cells to activate a protective response. We first describe how to generate hairpin RNAi constructs for constitutive genetic knockdown in specific tissues and how to perform tissue-specific genetic knockdown by feeding RNAi at different life stages. Stress reporters&#160;and behavioral assays function as&#160;valuable readouts&#160;that enable&#160;the fast screening of genes and conditions modifying systemic stress signaling processes. Finally, proteostasis sensors expressed in different tissues are utilized to determine changes in the tissue-specific capacity of the PN at different stages of development and aging. Thus, these tools should help clarify and allow monitoring the capacity of PN in specific tissues, while helping to identify components that function in different tissues to mediate cell-nonautonomous PN in an organism.

Maintenance of organismal proteostasis requires the coordination of protein quality control responses such as chaperone expression from one tissue to another. Here, we provide tools used in C. elegans that allow monitoring of proteostasis capacity in specific tissues and determine intercellular signaling responses.

Over the past decade there has been a transformative increase in knowledge surrounding the regulation of protein quality control processes, unveiling the ...

חוקר את החלבונים כמו פריון-הפצת ורעילות של שימוש במטזואניים דגם אורגניזם

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אוטומטיות התנהגותית ניתוח של גדול C. elegans אוכלוסיות שימוש רחב שדה-of-view מעקב פלטפורמה

ניטור-לתא שידור של אגרגטים חלבון פריאון-כמו בדרוזופילה Melanogaster

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

הקרנה תפוקה גבוהה של חלבון מבוססת ירושה cerevisiae ס

מדידת רעילות ב -C. אלגיה

כימות ירידה פרוטאוסטטית ספציפית לרקמות ב elegans Caenorhabditis

דוגמנות גיל הקשורים מחלות ניווניות בתוך האלאים

Tissue-Specific RNAi Tools to Identify Components for Systemic Stress Signaling