We thank Chaniotakis M. and Kounakis K. for video recording and editing. K.P. is funded by a grant from the Hellenic Foundation for Research and Innovation (HFRI) and the General Secretariat for Research and Technology (GSRT). N.T. is funded by grants from the European Research Council (ERC &#8211; GA695190 &#8211; MANNA), the European Commission Framework Programmes, and the Greek Ministry of Education.

1. Necrotic cell death-induced by hyperactive ion channels
NOTE: Gain-of-function mutations in the gene family of degenerins, including mec-4 and deg-3 among others, results in the generation of hyperactive ion channels triggering necrotic cell death of six touch receptor neurons required for mechanosensation in worms3. Necrosis induced by the aberrant stimulation of degenerins displays several mechanistic and morphological similarities to excitotoxicity in mammals. The maintenance of energy metabolism and calcium homeostasis has a crucial role on neuronal survival during necrosis11. The following strains can be used to monitor necrotic cell death triggered by hyperactive ion channels, mec-4(u231)X and deg-3(u662)V.
<ol>
	<li>Maintenance, synchronization and preparation of mutant worms for examination of necrotic cell death
	<ol>
		<li>Day 1: Pick L4 larvae of mec-4(u231) or deg-3(u662) mutant nematodes onto Nematode Growth Media (NGM; Table 1) plates seeded with Escherichia coli (OP50) using a dissecting stereomicroscope.</li>
		<li>Place 10 L4 nematodes per seeded NGM plate and grow them at the standard temperature of 20 &#176;C.</li>
		<li>Day 5: Wash the plates with 1 mL of M9 buffer (Table 1) and collect the animals in a 1.5 mL tube.</li>
		<li>Centrifuge at 10,000 x g for 30 s and remove the supernatant.</li>
		<li>Add 0.5 mL of bleaching solution (7 mL of H2O, 1 mL of 5 N NaOH and 2 mL of bleach). 
		NOTE: Bleaching solution gradually lose its efficiency; hence it should be prepared daily.</li>
		<li>Vortex and monitor periodically until the worms have dissolved. 
		NOTE: Avoid bleaching for periods longer than 5 min because the embryos&#8217; viability will be affected.</li>
		<li>Centrifuge at 10,000 x g for 30 s and remove the supernatant.</li>
		<li>Wash twice the pellet (eggs) with 1 mL of M9 buffer.</li>
		<li>After washing, resuspend the eggs in 200 &#181;L of M9 buffer and incubate them for 25 min at 34 &#176;C in a water bath.</li>
		<li>Maintain a separate group of control sample (eggs) at 20 &#176;C.</li>
		<li>Pipette 100 &#181;L of control or heat shock-treated eggs and place them on unseeded NGM plates. Each plate contains at least 100-200 eggs.</li>
		<li>Incubated the eggs at 20 &#176;C until hatching.</li>
	</ol>
	</li>
	<li>Mounting L1 larvae for examination using differential interference contrast (DIC; Nomarsky) microscopy
	<ol>
		<li>Prepare 2% agarose pads.</li>
		<li>Use 1 mL of M9 buffer to wash the plates and collect L1 larvae nematodes in 1.5 mL tubes.</li>
		<li>Centrifuge at 10,000 x g for 30 s.</li>
		<li>Remove the supernatant and keep the pellet (nematodes).</li>
		<li>Add 100 &#181;L of 20 mM M9/levamisole buffer to anesthetize the nematodes. 
		NOTE: Avoid sodium azide as an anesthetic. Sodium azide triggers mitochondrial damage and oxidative stress leading eventually to cell death induction</li>
		<li>Pipette 10 &#181;L of M9/levamisole buffer containing L1 worms and mount them on 2% agarose pads (Table 1).</li>
		<li>Gently place a coverslip on the top of the sample. 
		NOTE: Seal the coverslip with nail polish to preserve humidity throughout the process of imaging.</li>
		<li>Observe worms using differential interference contrast (DIC; Nomarsky) microscopy.</li>
		<li>Score neurodegeneration of the six touch receptor neurons by counting cells with the characteristic vacuolated appearance per nematode.</li>
	</ol>
	</li>
</ol>
2. Protein aggregate-induced neurodegeneration
NOTE: The following strains can be used to investigated protein aggregates-induced neurotoxicity: (A) overexpression of human &#945;-synuclein in dopaminergic neurons, UA44: Is[baIn1; pdat-1&#945;-syn, pdat-1GFP] and (B) overexpression of human polyglutamine protein (PolyQ) pan-neuronally, AM101: rmsIs110[prgef-1Q40::YFP]6,10.
<ol>
	<li>Maintenance, synchronization and preparation of transgenic nematodes for neurodegeneration assay
	<ol>
		<li>Use a dissecting stereomicroscope to monitor C. elegans development and growth.</li>
		<li>Day 1: Synchronize the population of transgenic nematodes by picking and transferring 15-20 L4 larvae of each strain on freshly OP50-seeded NGM plates. 
		NOTE: Use at least three plates of each strain per condition.</li>
		<li>Incubate and let the nematodes to grow at the standard temperature of 20 &#176;C.</li>
		<li>Day 2: Perform daily preconditioning for 30 minutes by transferring the plates in an incubator set at 34 &#176;C. Then, return the preconditioned nematodes back at the standard temperature of 20 &#176;C. 
		NOTE: Different genetic backgrounds might be sensitive to high temperature for long periods exposure.</li>
		<li>(A) If investigating overexpression of human &#945;-synuclein in dopaminergic neurons, UA44: Is[baIn1; pdat-1&#945;-syn, pdat-1GFP]: on Day 9, monitor 7-days-old transgenic nematodes for dopaminergic neuronal cell death.</li>
		<li>(B) If investigating overexpression of human polyglutamine protein (PolyQ) pan-neuronally, AM101: rmsIs110[prgef-1Q40::YFP]: on Day 5, measure neuronal PolyQ aggregates in the head region of 4-days-old transgenic animals expressing Q40::YFP.</li>
	</ol>
	</li>
	<li>Mounting the samples for microscopic examination
	<ol>
		<li>Prepare 2% agarose pads (Table 1).</li>
		<li>Add 10 &#181;L of 20 mM M9/levamisole buffer drop at the center of the agarose pad.</li>
		<li>Pick the respective transgenic nematodes and transfer them in a M9/levamisole drop. 
		NOTE: Place 20-30 nematodes per drop.</li>
		<li>Place gently a coverslip on the top of the sample. 
		NOTE: Seal the coverslip with nail polish to preserve humidity throughout the process of imaging.</li>
		<li>Proceed to microscopic examination of the respective samples.</li>
	</ol>
	</li>
	<li>Acquisition process and data analysis of transgenic nematodes co-expressing &#945;-synuclein and cytosolic GFP in dopaminergic neurons
	<ol>
		<li>Use an epifluorescence microscope combined with a camera (e.g., EVOS FL Auto 2).</li>
		<li>Detect and capture z-stack images of the head region at 20x magnification.</li>
		<li>Save and collect the maximum intensity projection images.</li>
		<li>Proceed to the analysis of the acquired images.</li>
		<li>Examine the transgenic worms for neurodegeneration by scoring the following cellular characteristics, (i) loss of fluorescence from neurons expressing GFP under the promoter of dat-1, (ii) neurons showing soma and/or axonal blebbing, outgrowths or dendritic loss.</li>
		<li>Import and analyze the data by using a software package (e.g., Excel).</li>
	</ol>
	</li>
</ol>
3. Acquisition process and data analysis of transgenic nematodes expressing pan-neuronally PolyQ40 fused with YFP
<ol>
	<li>Use an epifluorescence microscope combined with a camera (e.g., EVOS FL Auto 2).</li>
	<li>Detect and capture z-stack images of the head region at 20x magnification.</li>
	<li>Save and collect the maximum intensity projection images.</li>
	<li>Proceed to the analysis of the acquired images by using Fiji software.</li>
	<li>Open images with Fiji program14.</li>
	<li>Select Split Channels command via the Image and Color drop-down menu. 
	NOTE: Keep the green channel image.</li>
	<li>Use the freehand selection tool to manually set the fluorescent region of interest (ROI; e.g., head).</li>
	<li>Add the respective ROI in ROI Manager via Analyze and Tools drop-down menu.</li>
	<li>Subtract the background to 50% by selecting Process and Subtract Background.</li>
	<li>Set up and apply threshold values via the menu command Image | Adjust | Threshold. Keep and set the same threshold values throughout image analysis of the entire experiment.</li>
	<li>Select the respective ROI from the ROI Manager.</li>
	<li>Analyze the number of protein aggregates by using the menu command Analyze and Analyze Particles.</li>
	<li>Repeat steps 3.5-3.12 for each acquired image.</li>
	<li>Copy the display values from the separate Results window.</li>
	<li>Paste/Import and analyze the results by using a software package.</li>
</ol>
4. Report statistical analysis
<ol>
	<li>Use at least 30 nematodes for each experimental condition. Perform three biological replicates.</li>
	<li>Use statistical analysis software to either conduct student t-test (comparison between two groups) or ANOVA (comparison among multiple groups) for statistical analysis with p &#60; 0.05 as significant.</li>
</ol>

<ol><li>Nikoletopoulou, V., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Necrotic+cell+death+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">Necrotic cell death in Caenorhabditis elegans.</a> Methods in Enzymology. 545, 127-155 (2014).</li>
<li>Syntichaki, P., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+biochemistry+of+neuronal+necrosis%3A+rogue+biology%5BTitle%5D)+AND+%22Nature+Reviews+Neuroscience%22%5BJournal%5D)">The biochemistry of neuronal necrosis: rogue biology.</a> Nature Reviews Neuroscience. 4 (8), 672-684 (2003).</li>
<li>Syntichaki, P., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+models+of+mechanotransduction%3A+the+nematode+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Physiological+Reviews%22%5BJournal%5D)">Genetic models of mechanotransduction: the nematode Caenorhabditis elegans.</a> Physiological Reviews. 84 (4), 1097-1153 (2004).</li>
<li>Berkowitz, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+a+C.+elegans+dopamine+neuron+degeneration+assay+for+the+validation+of+potential+Parkinson%27s+disease+genes%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Application of a C. elegans dopamine neuron degeneration assay for the validation of potential Parkinson's disease genes.</a> Journal of Visualized Experiments. (17), (2008).</li>
<li>Brignull, H. R., Moore, F. E., Tang, S. J., Morimoto, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polyglutamine+proteins+at+the+pathogenic+threshold+display+neuron-specific+aggregation+in+a+pan-neuronal+Caenorhabditis+elegans+model%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Polyglutamine proteins at the pathogenic threshold display neuron-specific aggregation in a pan-neuronal Caenorhabditis elegans model.</a> Journal of Neuroscience. 26 (29), 7597-7606 (2006).</li>
<li>Gidalevitz, T., Ben-Zvi, A., Ho, K. H., Brignull, H. R., Morimoto, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progressive+disruption+of+cellular+protein+folding+in+models+of+polyglutamine+diseases%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Progressive disruption of cellular protein folding in models of polyglutamine diseases.</a> Science. 311 (5766), 1471-1474 (2006).</li>
<li>Gitler, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Parkinson%27s+disease+protein+alpha-synuclein+disrupts+cellular+Rab+homeostasis%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">The Parkinson's disease protein alpha-synuclein disrupts cellular Rab homeostasis.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (1), 145-150 (2008).</li>
<li>Klaips, C. L., Jayaraj, G. G., Hartl, F. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathways+of+cellular+proteostasis+in+aging+and+disease%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">Pathways of cellular proteostasis in aging and disease.</a> Journal of Cell Biology. 217 (1), 51-63 (2018).</li>
<li>Labbadia, J., Morimoto, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+biology+of+proteostasis+in+aging+and+disease%5BTitle%5D)+AND+%22Annual+Review+of+Biochemistry%22%5BJournal%5D)">The biology of proteostasis in aging and disease.</a> Annual Review of Biochemistry. 84, 435-464 (2015).</li>
<li>Tucci, M. L., Harrington, A. J., Caldwell, G. A., Caldwell, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+dopamine+neuron+degeneration+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Modeling dopamine neuron degeneration in Caenorhabditis elegans.</a> Methods in Molecular Biology. 793, 129-148 (2011).</li>
<li>Kourtis, N., Nikoletopoulou, V., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+heat-shock+proteins+protect+from+heat-stroke-associated+neurodegeneration%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Small heat-shock proteins protect from heat-stroke-associated neurodegeneration.</a> Nature. 490 (7419), 213-218 (2012).</li>
<li>Kourtis, N., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+heat+shock+proteins+and+neurodegeneration%3A+recent+developments%5BTitle%5D)+AND+%22BioMolecular+Concepts%22%5BJournal%5D)">Small heat shock proteins and neurodegeneration: recent developments.</a> BioMolecular Concepts. 9 (1), 94-102 (2018).</li>
<li>Kumsta, C., Chang, J. T., Schmalz, J., Hansen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hormetic+heat+stress+and+HSF-1+induce+autophagy+to+improve+survival+and+proteostasis+in+C.+elegans%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Hormetic heat stress and HSF-1 induce autophagy to improve survival and proteostasis in C. elegans.</a> Nature Communications. 8, 14337(2017).</li>
<li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiji%3A+an+open-source+platform+for+biological-image+analysis%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li>
<li>Palikaras, K., Lionaki, E., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coordination+of+mitophagy+and+mitochondrial+biogenesis+during+ageing+in+C.+elegans%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans.</a> Nature. 521 (7553), 525-528 (2015).</li>
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</ol>

The authors declare no competing interests.

Here, we introduce and describe widely accessible methodologies for growth, synchronization and microscopic examination of some versatile C. elegans models investigating age-dependent neurodegeneration. Particularly, we assess and dissect the cellular and molecular underpinnings of age-related neuronal breakdown by using hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity1,2,3,</...

Over the last two decades, C. elegans has been widely used as a model organism to investigate the molecular mechanisms of necrotic cell death. C. elegans offers an exceptionally well-characterized and mapped nervous system, transparent body structure and a diverse repertoire of genetic and imaging methods to monitor in vivo cellular function and survival throughout ageing. Thus, several C. elegans genetic models of neurodegeneration have been already developed to assess neuronal viability. In particular, well-described and used nematode models include the hyperactive ion channel-induced necrosis1,2,3 and cell death triggered by increased protein aggregation4,5,6,7,8,9,10 and heat stroke11,12, among others.
Short-term exposure to sub-lethal temperatures conferred resistance against necrotic cell death, triggered by a subsequent heat stress both in nematodes and mammalian neurons11. Interestingly, daily preconditioning of nematodes at a mild elevated temperature protects against neurodegeneration, which is inflicted by diverse stimuli, such as ionic imbalance (e.g., mec-4(u231) and/or deg-3(u662)) and protein aggregation (e.g., &#945;-synuclein and polyQ40)11,13.
Here, we describe versatile methodologies using C. elegans to monitor and evaluate age-dependent neurodegeneration in well-established models of human diseases, such as excitotoxicity-triggered cell death, Parkinson&#8217;s and Huntington&#8217;s disease. Moreover, we underline the neuroprotective role of heat preconditioning in several models of neurodegeneration. A combination of these techniques together with genetic and/or pharmacological screens will result in the identification and characterization of novel cell death modulators, with potential therapeutic interest.

<table><tbody><tr><td>Agar</td><td>Sigma-Aldrich</td><td>5040</td><td></td></tr><tr><td>Agarose</td><td>Biozym</td><td>8,40,004</td><td></td></tr><tr><td>AM101: &lt;em&gt;rmsIs110&lt;/em&gt;[p&lt;sub&gt;&lt;em&gt;rgef-1&lt;/em&gt;&lt;/sub&gt;Q40::YFP]</td><td>Caenorhabditis Genetics Center (CGC)</td><td></td><td></td></tr><tr><td>Calcium chloride dehydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;?2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>C5080</td><td></td></tr><tr><td>Cholesterol</td><td>SERVA Electrophoresis</td><td>17101.01</td><td></td></tr><tr><td>&lt;em&gt;deg-3(u662)V&lt;/em&gt; or &lt;em&gt;deg-3(d)&lt;/em&gt;</td><td>Caenorhabditis Genetics Center (CGC)</td><td></td><td>Maintain animals at 20 &amp;deg;C</td></tr><tr><td>DIC microscope (Nomarsky)</td><td>Zeiss</td><td></td><td>Axio Vert A1</td></tr><tr><td>Dissecting stereomicroscope</td><td>Nikon Corporation</td><td></td><td>SMZ645</td></tr><tr><td>Epifluorescence microscope</td><td>Thermo Fisher Scientific</td><td></td><td>EVOS Cell Imaging Systems</td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; OP50 strain</td><td>Caenorhabditis Genetics Center (CGC)</td><td></td><td></td></tr><tr><td>Greiner Petri dishes (60 mm x 15 mm)</td><td>Sigma-Aldrich</td><td>P5237</td><td></td></tr><tr><td>image analysis software</td><td>Fiji</td><td></td><td>https://fiji.sc</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>EMD Millipore</td><td>1,37,010</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>EMD Millipore</td><td>1,04,873</td><td></td></tr><tr><td>Magnesium sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>M7506</td><td></td></tr><tr><td>&lt;em&gt;mec-4(u231)X&lt;/em&gt; or &lt;em&gt;mec-4(d)&lt;/em&gt;</td><td>Caenorhabditis Genetics Center (CGC)</td><td></td><td>Maintain animals at 20 &amp;deg;C</td></tr><tr><td>Microscope slides (75 mm x 25 mm x 1 mm)</td><td>Marienfeld, Lauda-Koenigshofen</td><td>10 006 12</td><td></td></tr><tr><td>Microscope cover glass (18 mm x 18 mm)</td><td>Marienfeld, Lauda-Koenigshofen</td><td>01 010 30</td><td></td></tr><tr><td>Microsoft Office 2011 Excel software package</td><td>Microsoft Corporation, Redmond, USA</td><td></td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>EMD Millipore</td><td>1,06,586</td><td></td></tr><tr><td>Nematode growth medium (NGM) agar plates</td><td></td><td></td><td></td></tr><tr><td>Nystatin stock solution</td><td>Sigma-Aldrich</td><td>N3503</td><td></td></tr><tr><td>Peptone</td><td>BD, Bacto</td><td>211677</td><td></td></tr><tr><td>Phosphate buffer</td><td></td><td></td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>EMD Millipore</td><td>1,06,40,41,000</td><td></td></tr><tr><td>Standard equipment for preparing agar plates (autoclave, Petri dishes, etc.)</td><td></td><td></td><td></td></tr><tr><td>Standard equipment for maintaining worms (platinum wire pick, incubators, etc.)</td><td></td><td></td><td></td></tr><tr><td>statistical analysis software</td><td>GraphPad Software Inc., San Diego, USA</td><td></td><td>GraphPad Prism software package</td></tr><tr><td>Streptomycin</td><td>Sigma-Aldrich</td><td>S6501</td><td></td></tr><tr><td>Tetramisole hydrochloride</td><td>Sigma-Aldrich</td><td>L9756</td><td></td></tr><tr><td>UA44: &lt;em&gt;Is&lt;/em&gt;[baIn1; p&lt;sub&gt;dat-1&lt;/sub&gt;&amp;alpha;-syn, p&lt;sub&gt;dat-1&lt;/sub&gt;GFP]</td><td></td><td></td><td>Upon request: G. Caldwell (University of Alabama, Tuscaloosa AL)</td></tr></tbody></table>

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.

Necrotic cell death-induced by hyperactive ion channels 
Using the procedures presented here, mec-4(u231) and deg-3u662) mutant embryos were either incubated for 25 min at 34 &#176;C or kept at the standard temperature of 20 &#176;C. Upon hatching, the number of neuronal cell corpses was determined at the L1 larval stage of both groups. Necrotic cell death is diminished in nematodes that hatched from heat shock preconditioned eggs (Figure 1A-1B...

Watch this Scientific Journal Video about Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans at JoVE.com

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.

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 ...

modeling-age-associated-neurodegenerative-diseases-caenorhabditis

Research

JoVE Journal

Biology

5.4K Views.  Foundation for Research and Technology-Hellas, Greece. In de afgelopen decennia, de levensduur van de mens is verlengd, als gevolg van verbeteringen in de levensstandaard en de vooruitgang in de medische wetenschap. Leeftijd is de grootste risicofactor voor veel levensbedreigende ziekten, waaronder neurodegeneratieve aandoeningen, zoals Alzheimer, Parkinson en de Ziekte van Huntington. Hier demonstreren we methodologieën met behulp van een aantal veelzijdige nieuwe methodemodellen, waaronder hyperactieve ionenkanaal geïnduceerde necrose en eiwi...

Video: Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

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.

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 ...

Neuroscience

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.

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

Developmental Biology

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

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.

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

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 ...

Evaluation of Motor Impairment in C. elegans Models of Amyotrophic Lateral Sclerosis

The neurodegenerative disease amyotrophic lateral sclerosis (ALS) features progressive loss of motor neurons accompanied by muscle weakness and motor impairment that worsens with time. While considerable advances have been made in determining genetic drivers of ALS for a subset of patients, the majority of cases have an unknown etiology. Further, the mechanisms underlying motor neuron dysfunction and degeneration are not well understood; therefore, there is an ongoing need to develop and characterize representative models to study these processes. Caenorhabditis elegans can adapt their movement to the physical constraints of their surroundings, with two primary movement paradigms studied in a laboratory environment- crawling on a solid surface and swimming in liquid. These represent a complex interplay between sensation, motor neurons, and muscles. C. elegans models of ALS can exhibit impairment in one or both of these movement paradigms. This protocol describes two sensitive assays for evaluating motility in C. elegans: an optimized radial locomotion assay measuring crawling on a solid surface and an automated method for tracking and analyzing swimming in liquid (thrashing). In addition to the characterization of baseline motor impairment of ALS models, these assays can detect suppression or enhancement of the phenotypes from genetic or small molecule interventions. Thus, these methods have utility for studying ALS&#160;models and any C. elegans strain that exhibits altered motility.

Evaluation of Motor Impairment in C. elegans Models of Amyotrophic Lateral Sclerosis

This protocol describes two sensitive assays for discriminating among mild, moderate, and severe motor impairment in C. elegans models of amyotrophic lateral sclerosis, with general utility for C. elegans strains, with altered motility.

The neurodegenerative disease amyotrophic lateral sclerosis (ALS) features progressive loss of motor neurons accompanied by muscle weakness and motor ...

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million people worldwide. Since many details about PD etiology remain unknown, studies investigating genetic and environmental contributors to PD are needed to discover methods of prevention, management, and treatment. Proper characterization of dopaminergic neuronal loss may be relevant not only to PD research, but to other increasingly prevalent neurodegenerative disorders.
There are established genetic and chemical models of dopaminergic neurodegeneration in the Caenorhabditis elegans model system, with easy visualization of neurobiology supported by the nematodes&#39; transparency and invariant neuronal architecture. In particular, hermaphroditic C. elegans&#39; dopaminergic neuron morphological changes can be visualized using strains with fluorescent reporters driven by cell-specific promotors such as the dat-1 dopamine transporter gene, which is expressed exclusively in their eight dopaminergic neurons.
With the capabilities of this model system and the appropriate technology, many laboratories have studied dopaminergic neurodegeneration. However, there is little consistency in the way the data is analyzed and much of the present literature uses binary scoring analyses that capture the presence of degeneration but not the full details of the progression of neuron loss. Here, we introduce a universal scoring system to assess morphological changes and degeneration in C. elegans&#39; cephalic neuron dendrites. This seven-point scale allows for analysis across a full range of dendrite morphology, ranging from healthy neurons to complete dendrite loss, and considering morphological details including kinks, branching, blebs, and breaks. With this scoring system, researchers can quantify subtle age-related changes as well as more dramatic chemical-induced changes. Finally, we provide a practice set of images with commentary that can be used to train, calibrate, and assess the scoring consistency of researchers new to this method. This should improve within- and between- laboratory consistency, increasing rigor and reproducibility.

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

In this article, we showcase how to use a seven-point scoring system to consistently quantify changes to dopaminergic neuron dendrite morphology in C. elegans. This system is intended for analyses of dopaminergic neurodegeneration assays utilizing genetic, chemical, and age-based models of neurodegenerative disorders.

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million ...

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.

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

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

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

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

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

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

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.

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

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