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...

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+C.+elegans+dopamine+neuron+degeneration+assay+for+the+validation+of+potential+Parkinson's+disease+genes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyglutamine+proteins+at+the+pathogenic+threshold+display+neuron-specific+aggregation+in+a+pan-neuronal+Caenorhabditis+elegans+model.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+disruption+of+cellular+protein+folding+in+models+of+polyglutamine+diseases.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Parkinson's+disease+protein+alpha-synuclein+disrupts+cellular+Rab+homeostasis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathways+of+cellular+proteostasis+in+aging+and+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+proteostasis+in+aging+and+disease.">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. 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T., Schmalz, J., Hansen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormetic+heat+stress+and+HSF-1+induce+autophagy+to+improve+survival+and+proteostasis+in+C.+elegans.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+mitophagy+and+mitochondrial+biogenesis+during+ageing+in+C.+elegans.">Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans.</a> Nature. 521 (7553), 525-528 (2015).</li><li>Samara, C., Syntichaki, P., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+is+required+for+necrotic+cell+death+in+Caenorhabditis+elegans.">Autophagy is required for necrotic cell death in Caenorhabditis elegans.</a> Cell Death and Differentiation. 15 (1), 105-112 (2008).</li></ol>

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: rmsIs110[prgef-1Q40::YFP]</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dehydrate (CaCl2&#8729;2H2O)</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>deg-3(u662)V or deg-3(d)</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td>Maintain animals at 20 &#176;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>Escherichia coli 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>KH2PO4</td>
			<td>EMD Millipore</td>
			<td>1,37,010</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>EMD Millipore</td>
			<td>1,04,873</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate (MgSO4)</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>mec-4(u231)X or mec-4(d)</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td>Maintain animals at 20 &#176;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>Na2HPO4</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: Is[baIn1; pdat-1&#945;-syn, pdat-1GFP]</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 ...

Over the past decades, human lifespan is extended, due to improvements in standards of living and advances in medical science. Age is the biggest risk factor for many life-threatening diseases, including neurodegenerative disorders, such as Alzheimer's, Parkinson's and Huntington's disease. Here, we demonstrate methodologies utilizing some versatile new method models, including hyper-active ion channel induced necrosis and protein aggregates induces neurotoxicity, to monitor and dissect the cellular molecular mechanism of neural degeneration.Peak healthful larvae of make four and make three Newton nematodes onto nematode growth media plate, sieving with E.Coli using dissecting stereo microscope. Place ten and four nematodes, parasitic enzyme plate and grow them at the standard temperature, of 20 degree celsius. Five days later, wash the plate with 1 mL and 9 buffer and collect the animals in 1.5mL tube.Centrifuge at 30, 000 g for 30 seconds and remove the supernatant. Add 0.5 mL of plating solution. Plating solution is stored at the room temperature.Vortex and monitor periodically until they all have dissolved. Avoid plating for periods longer than five minutes. Centrifuge at 10, 000 g for 30 seconds and remove the supernatant.Wash twice the palette with 1 mL and nine buffers. Centrifuge at 10000 g for 30 seconds and remove the supernatant. After washing, resuspend the eggs in 200 microliters M9 buffer and incubate them for 25 minutes, at 34 degrees Celsius in a water bath.Maintain a separate group of eggs at 20 degree Celsius. Pipette 100 microliters containing control or heat shock treated eggs and place them on unseeded 10 GN plate. Each plate contains at least 100 to 200 eggs.Incubate the eggs at 20 degree celsius until hatching. Use and M9 buffer to wash plates and collect L1 life and nematodes in 1.5 ml tube. Centrifuge at 10, 000 g for 30 seconds.Remove the supernatant and keep the palette. Add 100 microliters of 20 millimolar M9 levangial buffer to anesthetize the nematodes. Pipette 10 microliters of M9 levangial buffer, containing L1 watch and mount them in 2%agar-ls spot place gently a coverslip on the top of the sample.Observe watch using the DIC microscopy. Scanning degeneration of the 630 receptor neurons by counting cells with a characteristic virtual aided appearance of that nematode. Synchronize nematodes by selecting and transferring 15 to 20 L4 larvae of each in strain on firstly bacteria C then GM plates.Incubate and let the nematodes to grow at the standard temperature of 20 degrees Celsius. Perform daily preconditioning for 30 minutes by transferring the plates in an incubator set at 34 degrees Celsius. Then return the preconditioned nematodes back at the standard temperature of 20 degrees Celsius.Add 10 microliters of 20 millimolar M9 levangial buffer drop at the center of the agar-ls spot. Pick the respective transgenic nematodes and transfer them in M9 levangial drop. Place 20 to 30 nematodes the per drop.Place gently cover slip on the top of the sample. Seal the cover slip with nail polish to preserve humidity throughout the imaging process. Using a fluorescence microscope combined with a camera, detect and capture your study images of the head region at 20 x magnification.Monitor seven days of transgenic nematodes for dopaminergic neuronal cell death. Measure the neuronal policul aggregates in the head region of four days old transgenic animals expressing 240 fusion with live people. Necrotic cell death induced a hyperactive ion channels is the mainest in nematodes that has former heat shock precondition eggs.Heat shock preconditioning, promoting a prediction against alpha-synuclein induced cell death in seven-day old adult hermaphrodite and decreases Q40 protein aggregates in the head region of four day old adults. Aging is a consequence of damage caused by gradual degeneration of cellular and tissue homeostasis. Thus understanding of manipulating age-related neuronal break down are top places priorities.The presented techniques combined with genetics and pharmacological screens could lead to a notification of normal cell death modulators with potential therapeutic interest.

modeling-age-associated-neurodegenerative-diseases-caenorhabditis

Research

JoVE Journal

Biology

5.2K Views.  Foundation for Research and Technology-Hellas, Greece. Negli ultimi decenni, la durata della vita umana è stata estesa, a causa del miglioramento del tenore di vita e dei progressi nella scienza medica. L'età è il principale fattore di rischio per molte malattie potenzialmente letali, tra cui i disturbi neurodegenerativi, come l'Alzheimer, il morbo di Parkinson e il morbo di Huntington. Qui, dimostriamo metodologie che utilizzano alcuni nuovi modelli di metodo versatili, tra cui necrosi indotta da canali ionici iperattivi e aggregati proteici ...