Name: quantifying levels of dopaminergic neuron morphological alteration and degeneration in caenorhabditis elegans
Uploaded: 2021-11-20T13:00:00.000+00:00
Duration: 6 min 17 s
Description: Watch this Scientific Journal Video about Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans at JoVE.com

We wish to acknowledge Ian T. Ryde, for contributions to the development of the scoring scale and for his support during the creation of this manuscript. This work was supported by the National Institutes of Health (T32ES021432 supported KSM, and P42ES010356 to JNM).

1. Prepare Worms for Imaging
NOTE: See related JoVE video article31:&#160;https://www.jove.com/v/835/&#160;
<ol>
	<li>For each experimental group, pipette or pick 20 to 30 worms to an imaging platform compatible with the imaging microscope. Most common platforms include 2% agarose gel pads mounted on glass slides with a coverslip31 and 96-well plates containing well volumes at or less than 100 &#181;L of liquid medium.</li>
	<li>Paralyze the worms by adding 30-90 mM sodium azide (NaN3), 2.5-8.5 mM levamisole HCl, or other paralyzing agent to the worms. If paralyzing in liquid, use a higher concentration of paralyzing agent than if paralyzing on agarose pads. Tap imaging platform gently to mix.</li>
	<li>Allow worms to paralyze completely. 
	NOTE: This may take several minutes.</li>
</ol>
2. Image Dopaminergic Neurons
<ol>
	<li>Locate worms&#39; head regions under single-color GFP fluorescence using imaging microscope capable of taking z-stacks.
	<ol>
		<li>Be mindful of exposure and aperture settings; avoid overexposure of dendrites and keep settings consistent across trials. Make the dendrites as bright as needed for clear visualization; this typically results in overexposure of the soma. 
		NOTE: Images included in this protocol were captured using 400x magnification.</li>
	</ol>
	</li>
	<li>Scroll through the focus to find upper and lower bounds where the dendrites are clear. Set these as upper and lower bounds for a z-stack image capture.</li>
	<li>Click to capture z-stack images for each worm. 
	&#8203;NOTE: All following steps may be performed at any time.</li>
</ol>
3. Prepare Dopaminergic Neuron Images for Scoring
<ol>
	<li>For each z-stack, open the image file using either the microscope software or an external image analysis software, load the stack in the software, and compress the stack into a single flattened image.</li>
	<li>Blind images between and within treatment groups manually or using automatic blinding software.</li>
</ol>
4. Score Dopaminergic Neuron Dendrites
<ol>
	<li>Work with one neuron image at a time. Choose one of the four CEP dendrites to assess for blebs, breaks, and irregularities including bends, kinks, and curves. Scoring from the left to right when the nose is at the top of the image is recommended to ensure repeatability in scoring.</li>
	<li>Using the following guidelines, assign one score value to the dendrite. See Figure 1 for representative scoring image examples. 
	0- no damage, &#34;perfect&#34; neurons 
	1- irregular (kinks, curves, etc.) 
	2- &#60;5 blebs 
	3- 5-10 blebs 
	4- &#62;10 blebs and/or breakage removing &#60;25% of total dendrite 
	5- breakage, 25-75% of dendrite removed 
	6- &#8203;breakage, &#62;75% dendrite removed
	<ol>
		<li>If multiple criteria are met within a single dendrite (i.e., kinks and blebs), assign the highest applicable score.</li>
		<li>Do not score dendrites that are not clearly visible, due to issues with image capture, overlapping with other dendrites, etc. If zooming in on a flattened z stack image, be mindful of enlarged pixels resembling false blebs.</li>
	</ol>
	</li>
	<li>Repeat for each dendrite. Repeat for all images.</li>
	<li>Record all scores. Scores may be un-blinded at this time.</li>
</ol>
5. Prepare and Present Data
<ol>
	<li>Calculate the total number of dendrites in each treatment group assigned to each neurodegeneration score. Calculate the total number of scored dendrites in each treatment group.</li>
	<li>Divide the neurodegeneration score tallies by the total number of dendrites scored in the treatment group. Present data as a proportion of dendrites within a treatment group at each neurodegeneration score.</li>
</ol>
6. Perform Statistical Analysis
<ol>
	<li>Using a programming software or manually, run a chi-squared test for independence between all treatment group pairs to be compared. When appropriate, apply a Bonferroni correction of the p-value according to the number of compared experimental groups to account for multiple comparisons. 
	NOTE: This test will determine significant differences between two groups, but details of the type of difference must be qualified by eye.
	<ol>
		<li>Select comparisons between experimental groups. This will vary based on experimental design. 
		&#8203;NOTE: In our experiments, typically, controls are compared to their respective treatment groups, all controls are compared, and all treatments are compared.</li>
	</ol>
	</li>
</ol>
7. Practice Scoring with Practice Set of Dopaminergic Neuron Images
<ol>
	<li>See Supplementary File 1 for a set of neuron images presenting across the full range of our scoring system with commentary and score key. This practice set is intended to train researchers new to this method and ensure inter-rater reliability.</li>
</ol>
8. Consider Alternate Protocol Options
<ol>
	<li>Instead of capturing z-stacks, complete scoring at the microscope, without saving or stacking images. 
	NOTE: This option reduces requirements for technology capabilities, but removes the option for creating an archive of neuron images to return to at a later time, requires manual blinding, and permits blinding only between and not within treatment groups.</li>
	<li>Instead of creating a single compressed image per stack, complete scoring by scrolling through the images of each z-stack. 
	NOTE: This option may be easier for some scorers and it reduces the risk for seeing false blebs on worms that moved during imaging and allows for scoring overlapping dendrites, but requires manual blinding and permits blinding only between and not within treatment groups.</li>
</ol>

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The authors have no disclosures.

This protocol demonstrates how to use the seven-point scale developed in our laboratory to quantify levels of dopaminergic neuron morphologic alteration and degeneration in C. elegans. We created and shared this scale as a tool to standardize analysis of dopaminergic neurodegeneration work in worms. Recognizing the importance of studying pathways involved in highly prevalent neurodegenerative diseases, many investigators take advantage of the C. elegans model&#39;s suitability for neurobiology visualization to study neurodegeneration29,31,32,33. However, there has yet to be an effort to reduce the large variation in how neuron damage is quantified across neurodegeneration research in worms. The scoring system presented here is thus intended to promote consistency in analyses and allow for comparison between studies.
Our scoring system may be used to analyze data derived from C. elegans experiments that use cell-specific fluorescent reporters that allow for visualization of dopaminergic neurons - specifically the CEP dendrites. Specifically, strains tagged at the dat-1 gene for GFP visualization of the dopaminergic neurons are compatible with this scoring protocol, though many other related transgenic models of PD do exist. It is possible that this scoring system would also be useful with those models; however, this should be validated prior to using them. In particular, it is possible (but not tested to our knowledge) strains with mCherry may not be well suited for this protocol as mCherry aggregation may be indistinguishable from blebs or lead to cell stress. Rather than providing a commentary on all specific models of PD and related neurodegenerative disorders, we focus on the scoring of neurodegeneration data itself. Additionally, this protocol focuses only on neuronal morphology and does not consider fluorescence levels of the soma. Neurodegeneration assays may be performed alongside behavioral assays relevant to neurodegenerative diseases, such as locomotion, lifespan, and health-span experiments. Levels of degeneration in established chemical, age-based, and genetic models of PD can also be confirmed and detailed using this scoring system. Measuring models, contributors, and pathways associated with PD and other neurodegenerative diseases can add to the scientific knowledge about these disorders and point towards how to manage the growing population of affected individuals. Having comparable neurodegeneration results across literature is key in supporting this goal.
To interpret the results derived from this scoring system, we propose considering each dendrite scored as n=1, because different neurons within the same worm often respond differently to treatment. This may be driven by the fact that only the CEPD neurons are directly exposed to the pseudocoelomic body fluid. As such, this allows the score spread of experimental groups to be displayed as proportions of the total number of dendrites scored in each group. This method, used for the representative results shown here, allows for easy comparison across treatment groups, accounts for differential responses within the same worm, and is easily analyzed with a Chi-squared test complimented by a Bonferroni correction for multiple comparisons. An example template for recording neuron scores and calculating percentages can be found in Supplementary File 2. We have considered two alternate methods for data analysis and identify flaws in each. The first option is averaging the scores of the four CEP neurons for each worm. This parametrizes the data; however, it assumes a linear relationship with increasing score and loses information about any variations in response to treatment within the same worm. The second option is to sum the scores of all four CEP neurons for each worm, which also parametrizes the data. This still assumes a linear relationship between scores, however it more capably accounts for differences within each worm than averaged scores by expanding the parameters of possible scores. However individual researchers decide to display their data, the results should be considered alongside experimental variables such as strain and worm age; for example, older worms have a higher expected baseline level of degeneration.
As these neurodegeneration score results are interpreted, researchers should also be aware of a few caveats and limitations of the scoring method. First, certain technological requirements are necessary to capture images suitable for scoring. The imaging microscope must support fluorescence channels and magnification and exposure settings that allow for clear visualization of CEP dendrites. As noted in the protocol, technological requirements may be reduced by protocol adjustments like scoring live images through the microscopic field rather than capturing images to be archived and scored at a later time. Second, possible statistical analysis methods for this data are limited as the data is non-parametric. The scoring scale is presumed to be progressive, but cannot be considered numeric since there are discrete score options and score increases are not necessarily proportional to each other with respect to biological function. For these reasons, chi-squared tests for independence are best suited for this type of data, meaning the statistical analysis depends on the observer to determine the direction of any statistical significance. Notably, the chi-squared test also only analyzes for differences in score distribution and is unable to provide evidence of differences in specific scoring categories. Finally, the functional significance of the morphological changes quantified by this scoring system have yet to be studied.
The future directions prompted by the development of this scoring system involve determining biological bases and correlations with individual neuron scores. Studying the functional significance (e.g. neuronal signaling, worm behavior) of all points on the scoring scale will inform how to better translate results to conclusions applicable to understanding the causes and consequences of neurodegenerative diseases and developing prevention and treatment options. Future research on neurodegeneration in worms should aim to discover connections to other morphology, such as worm shape and size. Additionally, neurodegeneration research can be supported by studying other reporter C. elegans strains to measure endpoints such as bioenergetics, reactive oxygen species production, and mitochondrial morphology.

Parkinson&#39;s disease (PD) is an increasingly common neurodegenerative disease affecting up to 10 million individuals worldwide1. Males and older individuals are at a higher risk for developing PD; the average age of onset for the disease is 60 years, and PD incidence climbs from a 0.3% incidence in the general population to 3% in individuals over 80 years of age1,2. Although the details of PD pathology are not fully understood, this progressive disorder involves the loss of dopaminergic neurons in the substantia nigra region of the midbrain. Hypothesized mechanisms of this neuronal loss involve mitochondrial dysfunction, oxidative stress, and inflammation2. The causes and risk factors for the disease are still being explored, but involve a combination of environmental and genetic factors1. For example, studies have found positive associations between lifelong pesticide use and PD, as well as genetic susceptibility to familial PD1,3.
The C. elegans model system, originally developed in part for neurobiology research4, is well suited for evaluating dopaminergic neuron loss in vivo. Nass and colleagues pioneered the use of C. elegans for dopaminergic neurodegeneration5, and many groups have since adopted the worm as a successful model for PD and dopaminergic dysfunction6,7,8,9,10,11,12,13,14,15,16,17,18,19,20. C. elegans are good neurodegenerative disease models for many of the same reasons that they are such a popular model organism for other areas of biology; their transparency allows for in vivo study of cellular processes, genetic manipulation in worms is relatively quick and easy, they have a short generation time of about three days, and they are easy to maintain21. Most PD worm models fall into one of three categories: age-based models, chemical models, and genetic models. The ability to synchronize a population of worms allows for the study of age-related neurodegeneration for an age based-model of neurodegenerative diseases associated with aging, such as PD22. Chemical exposures inducing PD-like neuronal defects have been established using a variety of chemicals including 6-hydroxydopamine (6-OHDA), rotenone, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)22. Worms are also successfully used as genetic models of PD; strains with select neural gene knockouts can model various neurodegenerative diseases1,4. Combinations of genetic and environmental factors, or &#34;gene-environment interactions,&#34; which likely play a major role in PD2,17,23,24,25,26,27,28, have been examined by several groups using C. elegans. Finally, age-related dopaminergic neurodegeneration has also been observed29,30. If using an appropriate neural transgenic strain in fluorescent imaging, any of these PD worm models may be used to study dopaminergic neurodegeneration.
Quantifying changes to neuronal morphology is a critical component of neurodegenerative research. In C. elegans, many fluorescent reporter strains have been used to visualize morphological changes and loss of neurons. Strains suitable for neuronal imaging feature a fluorescent protein associated with cell-specific promoters. For dopaminergic neurodegeneration assays, our laboratory has used the BY200 [vtIs1 (dat-1p::GFP, rol-6)] strain, which has a green fluorescent protein (GFP) tag in the dat-1 gene, expressed in the dopaminergic neurons. Note that the BY200&#39;s roller phenotype has a very low penetrance and is rarely observed. Other common strains used for this type of imaging include BY250 [dat-1p::GFP], BY273 [baEx18[dat-1p::GFP+dat-1p::WT &#945;-syn]], BZ555 [egIs1 [dat-1p::GFP]], and several others available from the Caenorhabditis Genetics Center (CGC) or upon request from specific laboratories1,21,22,29. These strains typically allow for visualization of all three classes of dopaminergic neurons: cephalic (CEP), anterior deirid (ADE), and postdeirid (PDE) neurons. C. elegans does not naturally express the alpha synuclein protein, but strains such as BY273 have been engineered to express it. However, we note that the scoring system we present was developed using BY200, which does not express alpha synuclein, and would need to be validated with that strain (or any other new strain) prior to use. Additional dopaminergic neurons are present in males but are rarely considered because males normally comprise &#60;1% of a C. elegans population. Here, we focus on the four CEP dopaminergic neurons found in the head region of C. elegans. This set of neurons is easily located under fluorescent microscopy, is present in both hermaphrodite and male worms, does not typically overlap with other areas of auto-fluorescence, and is commonly reported on in worm studies. Notably, though these neurons are not myelinated, the CEP dorsal (CEPD) neurons are directly exposed to the pseudocoelomic body fluid where as the CEP ventral neurons are not. A healthy set of CEP dendrites typically displays as relatively straight, uninterrupted lines. Degenerated dendrites may show any combination of irregularities and signs of damage, including pronounced dots called blebs along the line of the dendrite and breaks in the line of the dendrite. Examples of CEP neurons at varying levels of degeneration can be seen in Figure 1.
Although dopaminergic neurodegeneration is being studied by a growing number of C. elegans laboratories, there has been a large variation in analytical methods of quantifying dopaminergic neuron damage29,31,32,33,34. Many published studies have reported on the presence or absence of CEP soma with a binary scoring system of degenerative versus typical or wild type neurons31,32. These scoring methods can identify certain stressors that induce neurodegeneration but cannot quantify the details of the progression of more subtle neuronal damage, or easily detect differences between neurodegeneration as induced by unique chemicals or other variables. Additionally, scoring systems focused on the cell bodies may not be sensitive to less severe levels of damage or to neuronal damage affecting only part of the cell, such as the dendrite. Since the dendrite appears to have the largest range of consistently detectable morphologic changes in response to chemical stressors, we have selected them as the basis for our analysis. The scoring system we present here is modified from dendrite morphology based multi-point scales that have been previously used in our lab29,33. This system expands these five- and six-point scales into a seven-point scale to account for age-related morphological changes, such as higher expected numbers of kinks in older adult dendrites, and to differentiate between severe damage and complete dendrite loss. The purpose of introducing this scoring system is to provide the ability to capture a comprehensive picture of neurodegeneration at all levels of neuronal damage and provide a universal system to support consistency across C. elegan dopaminergic neurodegeneration research. Because scoring is inherently subjective, it is critical to maximize consistency between individuals scoring, and to blind the scorer to the identity of the images using manual blinding or an automatic blinding program35. To improve consistency, we present a series of training images and utilize JoVE&#39;s video capabilities to demonstrate our scoring system in detail. We recommend using a system that both permits automated blinded scoring and allows the scorer to quantify her or his scoring consistency by re-scoring a subset of images. This is particularly important when combining or comparing data from multiple scientists, or training scientists new to scoring.

<table><tbody><tr><td>96-well plate</td><td>VWR</td><td>29442-056</td><td>For imaging in wells</td></tr><tr><td>Blinder</td><td>Solibyte Solutions LLC</td><td></td><td>Free software that blinds between and within uploaded sets of image files</td></tr><tr><td>BY200 [vtIs1 (dat-1p::GFP, rol-6)]</td><td>Aschner Lab</td><td></td><td>&lt;em&gt;C. elegans&lt;/em&gt; strain suitable for dopaminergic neuron fluorescent imaging. May be subsituted by other strains with a fluorescent reporter driven by cell-specific promotors&lt;br/&gt; Available upon request from the Meyer lab</td></tr><tr><td>complete K-medium</td><td></td><td></td><td>51 mM sodium chloride, 32 mM potassium chloride, 3 mM calcium chloride, 3 mM magnesium sulfate, 13 mM cholesterol</td></tr><tr><td>Coverslips 22x22mm, No.1 glass</td><td>VWR VistaVision</td><td>48366-067</td><td>For imaging on slides</td></tr><tr><td>dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>472301</td><td>Solvent for rotenone exposures</td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td>ImageJ 1.5e or newer. Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https:// imagej.nih.gov/ij/, 1997-2016.</td><td>Sotware for image manipulation</td></tr><tr><td>Keyence BZ-X All-in-one Fluoresence Microscope</td><td>Keyence</td><td></td><td>Used for fluorescent dopaminergic neuron image capture. May be substituted by other microscopes stuitable for fluorescent, high-resolution imaging</td></tr><tr><td>Microscope Slides 3x1&quot;</td><td>VWR VistaVision</td><td>16004-420</td><td>For imaging on slides</td></tr><tr><td>Rotenone</td><td>Sigma-Aldrich</td><td>R8875</td><td>Electron transport chain complex I inhibitor</td></tr><tr><td>Sodium Azide (NaN_3)</td><td>Sigma-Aldrich</td><td>S2002</td><td>Paralytic</td></tr><tr><td>Sodium Hydroxide</td><td>Sigma-Aldrich</td><td>S2770</td><td>For bleach lysis</td></tr><tr><td>Sodium Hypochlorite</td><td>VWR</td><td>RC7495.5-32</td><td>For bleach lysis</td></tr><tr><td>Tetramisole (Levamisole) Hydrochloride (HCl)</td><td>Sigma-Aldrich</td><td>L9756</td><td>Paralytic</td></tr></tbody></table>

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.

The scoring system described here was used to assess neurodegeneration in L4 larval stage BY200 [vtIs1 (dat-1p::GFP, rol-6)] C. elegans after rotenone exposure. Results of this experiment are shown in Figure 2 and represent our scoring system&#39;s ability to detect and quantify variable levels of dopaminergic neuron damage. Rotenone is a naturally occurring electron transport chain complex I inhibitor used in some pesticides, piscicides, and insecticides36,37. Note that working with toxic chemicals such as rotenone is inherently dangerous, and all labs should comply with all use and disposal regulations set by their institutions. In this experiment, liquid rotenone exposures at two doses, 0.03 &#181;M and 0.5 &#181;M, along with a control group, were begun immediately following a 0.5 M sodium hydroxide/1% sodium hypochlorite lysis to harvest eggs38. Eggs hatched in complete K-medium33,39 with 0.25% dimethyl sulfoxide (DMSO), and worms remained in liquid for ~48 hours until mid-L4 larval stage, at which point they were removed from chemical exposure, and prepared, imaged, and, using Figure 1 as a reference, scored according to the protocol steps above. For the higher dose of 0.5 &#181;M rotenone, eggs were harvested 24 hours in advance to account for a rotenone-induced developmental delay and ensure all worms were stage synchronized at the time of imaging.
Figure 2&#160;further demonstrates how our laboratory visualizes data collected using this scoring system. In this figure, a dose-dependent neurodegeneration response can be appreciated, and the specific breakdown of the score distribution is displayed clearly. These particular results showcase how neuronal damage can present in different ways. For example, the 0.03 &#181;M rotenone-exposed group has a decreased proportion of healthy neurons with a score of 0, as compared to the control group, yet also has a decreased proportion of 5 scores. Detecting this detail about the score distributions between experimental groups highlights the sensitivity of our seven-point scoring system. This data was analyzed for statistical significance according to the protocol, using a chi-squared test for independence with a Bonferroni correction.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62894/62894fig01.jpg" /> 
Figure 1. Dopamine neuron morphological alteration and degeneration scoring system representative images. This consolidated chart contains examples of neurons at each score and is intended to be used as a reference for scoring. Here, each labelled score corresponds to the most damaged dendrite in each worm, as indicated by the arrow in each panel. These images were taken using the protocol described in this paper with BY200 C. elegans. Please see Supplementary File 1 for a set of scored images with commentary to be used for training those new to this scoring method. <a href="https://www.jove.com/files/ftp_upload/62894/62894fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62894/62894fig02.jpg" /> 
Figure 2. BY200 L4 dopamine neuron morphology and degeneration scores after rotenone exposure. This figure shows representative results analyzed using the scoring methods described here. The visualized greater proportions of damaged dopaminergic neurons with higher rotenone exposure concentrations were statistically analyzed using chi-squared tests for independence. Both rotenone treatment groups yielded statistically significant p-values when compared to the control group. Different letters indicate statistical difference. <a href="https://www.jove.com/files/ftp_upload/62894/62894fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1. <a href="https://www.jove.com/files/ftp_upload/62894/Supplemental_Training_Set_KM_V5.docx" target="_blank">Please click here to download this file.</a>
Supplementary File 2. <a href="https://www.jove.com/files/ftp_upload/62894/SupplementaryFile2.xlsx" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans at JoVE.com

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.

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 ...

quantifying-levels-dopaminergic-neuron-morphological-alteration

Research

JoVE Journal

Neuroscience

4.0K Views.  Duke University. 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. 

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

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

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 ...

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 ...

Examining the Effect of Pesticides on Caenorhabditis elegans Neurons

Caenorhabditis elegans is a powerful model organism used in many research laboratories to understand the consequences of exposure to chemical pollutants, pesticides, and a wide variety of toxic substances. These nematodes are easy to work with and can be used to generate novel research findings, even in the undergraduate biology laboratory. A multi-week laboratory series of authentic, student-driven research projects trains students in a toolkit of techniques and approaches in behavioral measurements, cell biology, and microscopy that they then apply to their projects. One technique in that toolkit is quantifying the percentage of neurons exhibiting neurodegenerative damage following exposure to a chemical toxicant like a pesticide. Young adult C. elegans nematodes can be exposed to different concentrations of commercially available pesticides or other types of toxicants for 2-24 h. Then, undergraduate students can visualize different neuron subtypes using fluorescent-expressing strains of C. elegans. These techniques do not require sophisticated image processing software and are effective at even low magnifications, making the need for expensive confocal microscopy unnecessary. This paper demonstrates how to treat the nematodes with pesticides and how to image and score the neurons. It also provides a straightforward protocol for the microscopy and analysis of neuron morphology. The materials used for this technique are inexpensive and readily available in most undergraduate biology departments. This technique can be combined with behavioral measures like locomotion, basal slowing, or egg-laying to conduct a potentially publishable series of experiments and give undergraduate students an authentic research experience at a very low cost.

Examining the Effect of Pesticides on Caenorhabditis elegans Neurons

Young adult Caenorhabditis elegans nematodes are exposed to different concentrations of commercial pesticides or other toxicants for 2-24 h. Then, different neurons can be visualized using fluorescent-expressing strains. This paper demonstrates how to expose nematodes to pesticides and assess neuron damage.

Caenorhabditis elegans is a powerful model organism used in many research laboratories to understand the consequences of exposure to chemical pollutants, ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel 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

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.

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

In Vivo Quantification of Protein Turnover in Aging C. Elegans using Photoconvertible Dendra2

Proteins are synthesized and degraded constantly within a cell to maintain homeostasis. Being able to monitor the degradation of a protein of interest is key to understanding not only its life cycle, but also to uncover imbalances in the proteostasis network. This method shows how to track the degradation of the disease-causing protein huntingtin. Two versions of huntingtin fused to Dendra2 are expressed in the C. elegans nervous system: a physiological version or one with an expanded and pathogenic stretch of glutamines. Dendra2 is a photoconvertible fluorescent protein; upon a short ultraviolet (UV) irradiation pulse, Dendra2 switches its excitation/emission spectra from green to red. Similar to a pulse-chase experiment, the turnover of the converted red-Dendra2 can be monitored and quantified, regardless of the interference from newly synthesized green-Dendra2. Using confocal-based microscopy and due to the optical transparency of C. elegans, it is possible to monitor and quantify the degradation of huntingtin-Dendra2 in a living, aging organism. Neuronal huntingtin-Dendra2 is partially degraded soon after conversion and cleared further over time. The systems controlling degradation are deficient in the presence of mutant huntingtin and are further impaired with aging. Neuronal subtypes within the same nervous system exhibit different turnover capacities for huntingtin-Dendra2. Overall, monitoring any protein of interest fused to Dendra2 can provide important information not only on its degradation and the players of the proteostasis network involved, but also on its location, trafficking, and transport.

In Vivo Quantification of Protein Turnover in Aging C. Elegans using Photoconvertible Dendra2

Presented here is a protocol to monitor degradation of the protein huntingtin fused to the photoconvertible fluorophore Dendra2.

Proteins are synthesized and degraded constantly within a cell to maintain homeostasis. Being able to monitor the degradation of a protein of interest is ...

Analyzing Mitochondrial Morphology in C. elegans

Imaging and Quantifying Mitochondrial Morphology in C. elegans During Aging

Mitochondria, important cellular organelles found in most eukaryotic cells, are major sites of energy production through aerobic respiration. Beyond this well-known role as the 'cellular powerhouse,' mitochondria are also involved in many other essential cellular processes, including the regulation of cellular metabolism, proliferation, immune signaling, and hormonal signaling. Deterioration in mitochondrial function during aging or under mitochondrial stress is often characterized by distinct changes in mitochondrial morphology and volume. The nematode C. elegans is an ideal model for studying these changes due to its transparent body and short lifespan, which facilitate live microscopy throughout its lifetime. However, even within the C. elegans field, numerous transgenic constructs and methods for mitochondrial imaging are available, each with its own limitations. Here, single-copy, matrix-localized GFP constructs are presented as a robust and reliable method for imaging mitochondrial morphology in C. elegans. This study specifically focuses on experimentally controllable factors to minimize errors and reduce variability between replicates and across studies when performing mitochondrial imaging during the aging process. Additionally, mitoMAPR is recommended as a robust method to quantify changes in mitochondrial morphology across tissue types during aging.

This protocol provides a standardized approach for imaging mitochondrial morphology in multiple tissues of C. elegans during aging.

Mitochondria, important cellular organelles found in most eukaryotic cells, are major sites of energy production through aerobic respiration. Beyond this ...

Studying the Effect of Pesticides on Caenorhabditis elegans Neurons

Source: Susman, K. M. R. <a href="https://app.jove.com/v/63845">Examining the Effect of Pesticides on Caenorhabditis elegans Neurons.</a> J. Vis. Exp. (2022).
This video demonstrates evaluating the neurodegenerative effects of a test chemical on Caenorhabditis elegans with fluorescently tagged neurons. Normal neurons show bright fluorescence of intact soma and processes, while diminished fluorescence, swollen soma, and punctuated processes indicate neurodegeneration.

1. Preparation of pesticide-coated Petri plates

	Prepare agar Petri dishes (6 cm diameter works best) using standard procedures.
	NOTE: These can be made ...

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

Summary

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