We acknowledge the following NIH grants: R01AG047101 and R37AG56510.&#160;Members of the Driscoll and Grant labs have contributed extensively to the development and fine tuning of protocols described, with rigorous experiments and strong communication.

<ol>
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The characterization of the in vivo molecular mechanisms of aggregate and organelle elimination in the form of large exophers is in its infancy. Questions as to the designation of cargoes for expulsion, the polarized collection of these cargoes within the cell, the regulation of the decision to generate exophers, the machinery that mediates extrusions, and the interaction of exophers with the degradative machinery in a neighboring cell all remain to be addressed. Furthermore, the in vivo visualization of tubular connections that can pass biological materials that include calcium, aggregates, and mitochondria is interesting and understudied biology in its own right. Questions of why certain cells are more prone to exopher production than others also are unresolved, but can begin to be genetically dissected with the approaches outlined in this protocol.
Described in detail in this protocol are the approaches to achieving reproducible scoring of exopher production, with attention to distinguishing exophers from nearby cell somas, timing of analyses to capture peak of exopher production, and strict control of growth conditions to eliminate unintended stresses that can modulate exopher levels. Both distinction of the large early exopher, or the &#8220;starry night&#8217; dispersion in the surrounding hypodermis can be quantitated as evidence of exopher production. That being said, neurons expressing mCherry under basal conditions are most often associated with 5-25% of neurons of a specific type producing an exopher. Controlled introduction of stress conditions could be applied to increase exopher production to detection as high as 90% of neurons producing extrusions, particularly useful for genetic or pharmacological screens for modifiers.
In human neurodegenerative disease, large aggregates can transfer from diseased neurons into neighboring cells to promote pathology spread. The exopher mechanism might transpire via a conserved mechanism used for aggregate extrusion across phyla. Defining the in vivo molecules that either enhance the efficiency of this process (considered more effective proteostasis control) or block it might be harnessed to influence design of novel strategies for combating multiple neurodegenerative diseases. As such, the protocol described here could be used for classical genetic mutagenesis screens, genome-wide RNAi screens that systematically knock down genes to identify enhancers and suppressors, or for drug intervention studies that identify candidate pharmacological modifiers of this process. The approach is straightforward, although somewhat laborious. Exophers are so large they can be viewed with a high-magnification dissecting microscope. Still, C. elegans neurons are relatively small and looking at their organelles or their membranes require higher power confocal images and is a slow process. Options for higher throughput could involve high content imaging approaches in multi-well plate format.
The application of a standardized approach to exopher scoring should underlie a concerted genetic dissection of the process by which neurons can organize and eliminate cellular debris.

The neurotoxic challenges of aggregates and dysfunctional mitochondria have long been considered to be cell-intrinsic, but more recently it has become clear that misfolded human disease proteins originating in one neuron can also spread to neighboring cells, promoting pathology1. Likewise, mammalian mitochondria can be sent out of the cell of their original production for transcellular degradation2 or for rescue of mitochondrial populations in challenged neighboring cells3. Vesicles of various sizes have generally been observed to transfer cellular materials to neighboring cells or to fluid surroundings4. Some extruded vesicles approach the size of the average neuronal soma (average touch neuron soma ~ 6 &#181;m) and can accommodate large aggregates and organelles.
A striking example of large vesicle extrusion that can carry protein aggregates and organelles occurs in C. elegans touch receptor neurons that express a high copy number reporter construct encoding a noxious aggregation-prone, degradation-resistant mCherry5. Extrusions from the touch neurons, called exophers, are ~4 &#181;m average diameter, selectively include mCherry or other aggregates, and are delivered directly into the neighboring hypodermis, which normally surrounds the touch receptor neurons. The hypodermis attempts lysosome-based degradation, but some non-digestible contents such as mCherry aggregates can be re-extruded by the hypodermis into the fluid-filled pseudocoelom of the animal, from which the mCherry can be taken up by remote scavenger cells called coelomocytes for long term storage (Figure 1, Figure 2)5.
The large extruded exopher vesicles leave the cell surrounded by touch receptor plasma membrane and can contain aggregated human disease proteins, mitochondria, and lysosomes. The process of exopher production appears to involve sorting of potentially toxic species (for example an aggregation-prone expressed mCherry is segregated from soluble, inoffensive proteins like GFP that remains mostly in the neuronal soma). In this way, directed expulsion of the threatening entities is accomplished by the neuron5. A proteostasis challenge, such as stress induced by&#160;autophagy knockdown, MG132-mediated proteasome inhibition, or transgenic expression of human disease proteins such as Huntington&#8217;s disease-associated expanded polyglutamine Q128 or Alzheimer&#8217;s disease-implicated fragment A&#946;1-42, can increase the numbers of neurons that produce exophers5.
As exophers have only recently been documented, what is known of their biology merits description. Exophers were discovered in, and are the most well studied in, C. elegans touch receptor neurons. There are six C. elegans mechanosensory touch neurons that have cell bodies distributed around the body (Figure 3A) and are called microtubule cells because their ultrastructure features distinctive 15 protofilament microtubules. The touch receptor neurons are the anterior AVM (anterior ventral microtubule neuron), ALMR, and ALML (anterior lateral microtubule neurons right and left), the more central PVM (posterior ventral microtubule neuron), and the posterior PLMR and PLML (posterior lateral microtubule neurons right and left) in the tail. Interestingly, the six touch receptor neurons produce exophers at different rates, despite expressing the same offensive transgene (Figure 3C). Of the six mechanosensory touch receptor neurons, the ALMR neuron undergoes exophergenesis more often than the other touch neurons. Quantitation of exopher numbers from touch neurons is thus usually established by focusing upon the ALMR.
Exophergenesis is a dynamic process that typically begins with swelling of the neuronal cytoplasm (Figure 1A-B). Cellular contents, organelles, or protein aggregates are collected to one side of the neuronal soma, most commonly toward the posterior end of the ALMR neuron (away from the projecting neurite), forming a pre-exopher domain (PED) (Figure 1B). The early protrusion is observed as the PED begins to project outwards, forming a recognizable protruded bud. The late bud is defined when the widest diameter of the pre-exopher domain is approximately &#8531; larger than the diameter of the constriction of the soma-exopher neck (Figure 1C).&#160;Exophers can be ejected in nearly any direction from the soma, but most exophers exit posteriorly from the cell body and remain in approximately the same focal plane as the originating soma.
The exopher can move away from the originating soma as the neck of the bud narrows into a thin filament. Exophers can remain attached to the soma via this filament (Figure 1D, arrow) and can later become detached. Cellular contents such as calcium, aggregates, and mitochondria can be transferred via this filament into the attached exopher5, although the bulk of extruded material is put into the exopher compartment by the massive budding event. Exophers are considered mature when there is no visible connecting tube or thin filament and the exopher is fully separated from the sending soma (Figure 1E).
Exophers produced by C. elegans touch neurons immediately encounter the hypodermis, the tissue that surrounds the touch neuron. Most commonly, the exopher vesicle appears to travel within the hypodermis posteriorly towards the tail, and can be fairly distant from the soma before exopher contents appear targeted for degradation (for example, the distance can be ~100 &#181;m away from the soma (Figure 1F)). The fluorescent exopher vesicle breaks up into many smaller vesicles within the hypodermis, taking on an appearance referred to as &#8220;starry night&#8221; (Figure 1G and Figure 2). In the &#8220;starry night&#8221; stage, punctate fluorescent material can be observed scattered across the hypodermal syncytium into many smaller points of fluorescence as compared to the original solitary exopher. Starry night can look punctate under low magnification and with higher magnification, can look punctate and/or networked within the hypodermis. The fluorescent signal of the starry night is typically dimmer than the exopher and the neuronally expressed fluorescence (Figure 2B-C). The dispersal of mCherry into many punctate vesicles is thought to involve phagosome maturation and fusion with the endosomal/lysosomal network of the hypodermal cell. Some exopher materials are likely degraded in the hypodermal lysosomal network, but residual species that are resistant to degradation (such as mCherry aggregates) are thrown out of the hypodermis into the pseudocoelom, a fluid compartment that can contain cellular debris. The fluorescent material is later taken in by remote scavenger cells called coelomocytes (Figure 2C), which can concentrate, store, and again attempt degradation of mCherry.
The phenomenon of aggregate extrusion and transfer appears conserved across phyla, having been reported in genetic models such as C. elegans5,6,7 and D. melanogaster8,9 as well as in multiple mammalian models. Exopher-like extrusions have been reported for mammalian cells10, an observation suggesting that conserved mechanisms might underlie aggregate and organelle expulsion. Exopher production may thus be a conserved mechanism of cellular debris management that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, which, when imbalanced, might actively contribute to neurodegenerative disease. Identification of the molecules involved in debris discrimination and sorting, transport to a distinct subcellular locale, extrusion, formation/scission of the tubular connection linking the soma and late exopher, and recognition of the large extruded vesicle for remote degradation by a neighboring cell remain for future work. Studies in nematode and fly models will be critically important to defining mechanisms of aggregate and organelle collection and transfer, utilizing unbiased genetic approaches and powerful cell biological tools offered by these models to identify participating molecules in physiological context.
Critical first steps in deciphering mechanisms operative in exopher biology involve defining protocols for reproducible in vivo exopher quantification. The C. elegans model offers a particular advantage for such efforts since the body is transparent and exophers can be readily observed when they contain fluorescently tagged proteins or organelles. Exophers have been reported to be generated by C. elegans dopaminergic neurons PDE and CEP, ASE and ASER sensory neurons, and dye-filling amphid neurons5. Because exophers produced by touch receptor neurons are best characterized, the focus here is on the use of touch neurons for exopher analysis. However the basic approach can be applied to measure exopher production from any cell. Protocols to detect and quantitate exophers produced by C. elegans touch receptor neurons that transgenically express mCherry protein are outlined, with an emphasis on cargoes that can be monitored and temporal constraints in scoring. This article defines approaches toward in vivo exopher identification, and the quantitation of environmental and genetic conditions that modulate exopher production. Protocols emphasize critical attention to constant non-stress conditions for the determination of baseline exopher production and for comparisons across genotypes.

<table><tbody><tr><td>95B Scientific CMOS camera</td><td>Photometrics Prime</td><td></td><td></td></tr><tr><td>1,000 &amp;mu;L low retention tips</td><td>Sarstedt</td><td></td><td></td></tr><tr><td>10 mL serological pipette</td><td>Appleton Woods</td><td>CC214</td><td></td></tr><tr><td>10 &amp;mu;L low retention tips</td><td>Sarstedt</td><td>70.1130.105</td><td></td></tr><tr><td>13% sodium hypochlorite</td><td>Acros Organics</td><td>AC219255000</td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td>Fisher Scientific</td><td>05-539-12</td><td></td></tr><tr><td>2 L erlenmeyer flasks</td><td>Scientific Laboratory Supplies</td><td>FLA4036</td><td></td></tr><tr><td>25 mL serological pipette</td><td>Appleton Woods</td><td>CC216</td><td></td></tr><tr><td>300 &amp;mu;L low retention tips</td><td>Sarstedt</td><td>70.765.105</td><td></td></tr><tr><td>50 mL serological pipette</td><td>Appleton Woods</td><td>CC117</td><td></td></tr><tr><td>5-Fluoro-2&#039;-deoxyuridine 98%</td><td>Alfa Aesar</td><td>L16497.ME</td><td></td></tr><tr><td>9 cm sterile Petri dishes</td><td>Fisher Scientific</td><td>11309283</td><td></td></tr><tr><td>absolute ethanol</td><td>Vwr</td><td>20821.33</td><td></td></tr><tr><td>Agar</td><td>Sigma Aldrich</td><td>A1296</td><td></td></tr><tr><td>C. elegans strain wild type</td><td>Supplied by CGC</td><td>N2</td><td>C. elegans strain</td></tr><tr><td>calcium chloride dihydrate</td><td>Sigma Aldrich</td><td>C3881</td><td></td></tr><tr><td>cholesterol</td><td>Acros</td><td>110190250</td><td></td></tr><tr><td>dibasic sodium phosphate</td><td>Sigma Aldrich</td><td>S3264</td><td></td></tr><tr><td>E. coli strain OP50</td><td>Supplied by CGC</td><td>Op50</td><td>E coli strain</td></tr><tr><td>FBS10 Standard microscope</td><td>Meyer Instruments</td><td>KSC 410-1-100-1</td><td>FBS10 Standard with Plate Base, 100/100 Trinocular Head and Flip zoom</td></tr><tr><td>glass pipette 270 mm</td><td>Fisherbrand</td><td>FB50255</td><td></td></tr><tr><td>Heraeus Multifuge X3R</td><td>Thermofisher scientific</td><td>75004515</td><td></td></tr><tr><td>Inoculating Spreaders</td><td>Fisher Scientific</td><td>11821741</td><td></td></tr><tr><td>LB medium capsules</td><td>MP biomedicals</td><td>3002-031</td><td></td></tr><tr><td>LDI &amp;ndash; Laser Diode Illuminator</td><td>89 North</td><td></td><td></td></tr><tr><td>levamisole</td><td>Sigma Aldrich</td><td>16595-80-5</td><td></td></tr><tr><td>M4 multipette</td><td>Eppendorf</td><td>4982000012</td><td></td></tr><tr><td>magnesium sulphate</td><td>Sigma Aldrich</td><td>M7506</td><td></td></tr><tr><td>monobasic potassium phosphate</td><td>Sigma Aldrich</td><td>P0662</td><td></td></tr><tr><td>Multitron Standard shaking incubator</td><td>Infors HT</td><td>INFO28573</td><td></td></tr><tr><td>Nalgene 1 L Centrifuge pots</td><td>Fisher Scientific</td><td>3120-1000</td><td></td></tr><tr><td>P10 pipette</td><td>Eppendorf Research Plus</td><td>3123000020</td><td></td></tr><tr><td>P1000 pipette</td><td>Eppendorf Research Plus</td><td></td><td></td></tr><tr><td>P200 pipette</td><td>Eppendorf Research Plus</td><td>3123000055</td><td></td></tr><tr><td>pipeteboy 2</td><td>VWR</td><td>612-0927</td><td></td></tr><tr><td>Polystyrene microbeads</td><td>Sigma Aldrich</td><td>MFCD00131491</td><td></td></tr><tr><td>RC5C plus floor mounted centrifuge</td><td>Sorvall</td><td>9900884</td><td></td></tr><tr><td>Reusable ringed cytology slides</td><td>ThermoFisher Scientific</td><td>22037242</td><td></td></tr><tr><td>SK4005 zdIs5[Pmec-4GFP]</td><td>contract Driscoll lab</td><td></td><td>GFP expressed in touch neurons</td></tr><tr><td>sodium chloride</td><td>Sigma Aldrich</td><td>13422</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Fisher Chemical</td><td>S/4880/53</td><td></td></tr><tr><td>Tactrol 2 Autoclave</td><td>Priorclave</td><td></td><td></td></tr><tr><td>Triton-X</td><td>Thermofisher scientific</td><td>28313</td><td></td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>9005-64-5</td><td></td></tr><tr><td>X-Light V2 Spinning Disk Confocal Unit</td><td>CrestOptics</td><td></td><td></td></tr><tr><td>ZB4065 bzIs166[Pmec-4mCherry]</td><td>contract Driscoll lab</td><td></td><td>mCherry expressed in touch neurons</td></tr><tr><td>ZB4067 bzIs167[Pmec-4mitogfp Pmec-4mCherry4]; igIs1[Pmec-7YFP Pmec-3htt57Q128::cfp lin-15+]</td><td>contract Driscoll lab</td><td></td><td>Q128 expressed in touch neurons</td></tr><tr><td>ZB4509 bzIs166[Pmec-4mCherry]; bzIs168[Pmec-7LMP-1::GFP]</td><td>contract Driscoll lab</td><td></td><td>mitoROGFP expressed in touch neurons</td></tr><tr><td>ZB4528 bzIs166[Pmec-4mCherry]; zhsEx17 [Pmec-4mitoLS::ROGFP]</td><td>contract Driscoll lab</td><td></td><td>autophagy marker expressed in touch neurons</td></tr><tr><td>ZEISS Axio Vert.A1</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

quantitative approaches for scoring in vivo neuronal aggregate and organelle extrusion in large exopher vesicles in c. elegans

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease across species. Although these neurotoxic challenges have long been considered to be cell-intrinsic, considerable evidence now supports that misfolded human disease proteins originating in one neuron can appear in neighboring cells, a phenomenon proposed to promote pathology spread in human neurodegenerative disease.
C. elegans adult neurons that express aggregating proteins can extrude large (~4 &#181;m) membrane-surrounded vesicles that can include the aggregated protein, mitochondria, and lysosomes. These large vesicles are called &#8220;exophers&#8221; and are distinct from exosomes (which are about 100x smaller and have different biogenesis). Throwing out cellular debris in exophers may occur by a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, relevant to processes by which aggregates spread in human neurodegenerative diseases.
While exophers have been mostly studied in animals that express high copy transgenic mCherry within touch neurons, these protocols are equally useful in the study of exophergenesis using fluorescently tagged organelles or other proteins of interest in various classes of neurons.
Described here are the physical features of C. elegans exophers, strategies for their detection, identification criteria, optimal timing for quantitation, and animal growth protocols that control for stresses that can modulate exopher production levels. Together, details of protocols outlined here should serve to establish a standard for quantitative analysis of exophers across laboratories. This document seeks to serve as a resource in the field for laboratories seeking to elaborate molecular mechanisms by which exophers are produced and by which exophers are reacted to by neighboring and distant cells.

Watch this Scientific Journal Video about Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans at JoVE.com

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

This protocol describes approaches for detection and quantitation of large aggregate and/or organelle extrusions (~4 &#181;m) produced by C. elegans cells in the form of membrane-bound exophers. We describe strains, growth conditions, scoring criteria, timing, and microscopy considerations needed to facilitate dissection of this debris expulsion mechanism.

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and ...

quantitative-approaches-for-scoring-vivo-neuronal-aggregate-organelle

Nanyang Technological University

Research

JoVE Journal

Neuroscience

7.4K Views.  Rutgers University. We karakteriseren de in vivo mechanismen van aggregaat en organelle extrusie van neuronen via een proces genaamd exophergenesis, slecht begrepen biologie die waarschijnlijk relevant is voor neurodegeneratieve ziekte pathologie verspreid. Deze benaderingen zijn noodzakelijk voor het bereiken van reproduceerbare scoren van geëxtrudeerde neuronale exophers en tonen een platform voor de mechanistische dissectie van cellulaire afval eliminatie via neuronale overdracht naar naburige cellen. Het de...

Video: Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a&#160;model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism&#39;s genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

Developmental Biology

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

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

Biochemistry

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

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 ...

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 ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Biology

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 ...

Measuring RAN Peptide Toxicity in C. elegans

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

Measuring RAN Peptide Toxicity in C. elegans

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

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

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

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