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

Summary

This protocol describes approaches for detection and quantitation of large aggregate and/or organelle extrusions (~4 µm) 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.

Abstract

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 µm) membrane-surrounded vesicles that can include the aggregated protein, mitochondria, and lysosomes. These large vesicles are called “exophers” 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.

Introduction

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 µm) 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 µm 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 autophagy knockdown, MG132-mediated proteasome inhibition, or transgenic expression of human disease proteins such as Huntington’s disease-associated expanded polyglutamine Q128 or Alzheimer’s disease-implicated fragment Aβ_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 ⅓ larger than the diameter of the constriction of the soma-exopher neck (Figure 1C). 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 µm 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 “starry night” (Figure 1G and Figure 2). In the “starry night” 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. elegans^5,6,7 and D. melanogaster^8,9 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.

Protocol

1. Strains useful for exopher detection

1. Select a strain that expresses fluorescent cargoes within the neurons of C. elegans to readily visualize exophers.
   NOTE: Table 1 lists strains that have been used to visualize exophers produced in touch receptor neurons^5,11,12. In principle, any cell or neuronal type can be tested for exopher production by using a cell or tissue specific promoter to drive expression of a fluorescent protein that aggregates or is otherwise selected for extrusion.
2. Alternatively, use a dye-filling assay to visualize exophers in the amphid head neurons, which are open to the environment and amenable to backfilling^5,13.

2. Growth media

1. Prepare standard nematode growth media (NGM) to culture strains according to standard methods^14,15.
   NOTE: Lack of food, or fluoro-deoxyuridine (FuDR), used commonly to block progeny production, and can dramatically affect exopher production. Keep the population continuously fed (avoid even short periods of bacterial food exhaustion) and maintain animals at a constant temperature.

3. Animal husbandry critical for consistent exopher production

1. Raise animals on consistent media and with consistent bacterial food sources. Animals must not run out of bacterial food, even for short periods of time since food limitation can dramatically change exopher production levels.
2. Keep media recipes and preparation uniform throughout a study.
   NOTE: Changing media can affect basal levels of exopher production. Agar batches can influence baseline exopher levels, so when supply lots change, make a note of the date. Throw out stock plates after two weeks to ensure healthy bacterial food and to prevent dried agar, which causes changes in agar osmolarity that influence exopher levels.
3. For basal conditions, keep animals at a constant temperature of 20 °C. Rearing animals at variable temperatures (even temporary changes in temperature) can cause variations in the timing of maximal exopher production.
   NOTE: Temperature variability is not limited to culture conditions. Temperatures variations during experiments or at the lab bench can be impactful. For example, temperatures within a microscope room should not differ dramatically from the culture incubator or lab bench.
4. Do not use pharmacological anti-fertility interventions because fertilized eggs are critical for early adult production of exophers.
   NOTE: Use of Fluoro-deoxyuridine (FuDR)¹⁶ or C22¹⁷, must be avoided. When performing life-span or old-aged animal experiments, age-synchronized populations should be maintained by physically removing adults from their smaller progeny by picking them onto fresh plates spread with bacteria rather than using common pharmacological anti-fertility interventions.
5. Do not use contaminated cultures; reinitiate experiments in the event of biological compromise of the population or the plate. Bacterial or fungal contamination can induce stresses and metabolic changes in animals and must be absent from experimental populations.
   1. To maximize reproducible results, maintain cultures for at least two healthy, well-fed, contamination-free generations at 20 °C before experimentation to avoid potential environmentally-induced epigenetic changes.

4. Age synchronization for exopher scoring by bleaching, sucrose flotation, or L4 larvae picking

1. Keep experimental populations the same biological age, as exopher detection patterns vary with adult age and comparison of animals of mixed age populations can confound results. Always ensure successful synchronization of experimental animal populations by checking for the “white crescent” vulva morphology at the L4 stage.
   NOTE: Generally, peak exopher production for C. elegans mechanosensory ALMR neurons occurs on adult day 2-3 (Figure 3D), as measured from days post L4 stage. Adult day 1 is 24 hours after the L4 larval stage that is distinguished by “white crescent” vulva morphology (Figure 5E).
2. Prepare synchronized egg populations by bleaching gravid adults.
   1. Collect gravid adults filled with eggs by washing animals growing on an NGM plate. To wash, flood the plate with 1 mL of M9 buffer, pipet up and down to collect liquid with suspended animals and pipet into a 1.5 mL microcentrifuge tube. Pellet animals by gravitational settling or gentle centrifugation with a mini centrifuge and remove the supernatant.
   2. Add 150 µL of 5M NaOH and 150 µL of 6% sodium hypochlorite (bleach) in 1 mL in H₂O and mix by inversion for approximately 5 minutes.
      NOTE: Fresh bleaching solution ensures that the animal cuticle can be disrupted for egg-harvest. Progress in disrupting the cuticle can be monitored under a dissecting microscope; adults should break and release eggs at the point that bleaching should be stopped.
   3. Gently centrifuge with a minicentrifuge tube for 20 s and remove the supernatant. Add 1 mL of M9 buffer and centrifuge again, leaving about 100 µL on top of the pellet.
   4. Repeat steps 4.2.3 twice to remove traces of bleach solution. 
   5. Resuspend the eggs in the remaining volume and transfer to a fresh seeded NGM plate. Adults will be lysed, but many viable eggs should be in the preparation.
3. Prepare synchronized populations by timed egg-lay. 
   1. Pick 20 gravid adults to a seeded NGM plate using standard transfer protocols¹⁴. 
   2. Allow animals to freely crawl and lay eggs for 1.5 h (mutant strains with low brood sizes may require the introduction of more adult animals).
   3. Remove all adult animals from the plate by picking, leaving the synchronized egg population behind. Check plates a few hours later to verify that no viable adults have been missed during adult removal. 
4. Prepare synchronized egg populations by sucrose flotation selection of eggs.
   1. Collect animals and eggs from five NGM plates upon which gravid animals have been laying eggs for at least 24 hours by flooding plates with M9 solution with 0.1% detergent (such as Tween 20 or Triton X-100) and collecting into a 15 mL tube. Pellet adults by gentle centrifugation at room temperature (2,000 x g for 30 s).
   2. Remove supernatant and wash animals in 15 mL of fresh M9 three times, discarding the supernatant after each wash, being sure to keep the pellet enriched in animals and eggs.
   3. Retain 2 mL of supernatant and resuspend the pellet. Add 2 mL of 60% weight by volume sucrose.
   4. Centrifuge at 2000 x g for 5 min. The solution will now display an upper phase highly enriched in eggs.
   5. Transfer about 2.5 mL of the upper phase to a new 15 mL tube and add 10 mL of M9.
   6. Mix by inversion for 1 min, and then centrifuge 2000 x g for 1 min.
   7. Remove the supernatant and wash the egg-enriched pellet in M9. 10-15 µL of the egg-pellet can be distributed to a fresh OP50-seeded NGM plate.
      NOTE: This method prepares a great number of eggs; do not allow collected animals to run out of OP50 E. coli food.
5. Prepare synchronized populations by picking animals at the L4 stage of development.
   1. Grow animals on seeded NGM plates as described above.
      NOTE: C. elegans develop in four discrete stages. At 20 °C, a newly laid egg takes about 9 hours to hatch (Figure 5A). Post hatch, an animal passes through larval stage 1 (L1) to larval stage 4 (L4), with each stage lasting 8-12 hours between each molt (Figure 5A-F). Therefore, a plate prepared by inoculating with eggs should have many L4 animals to pick about 40 hours after eggs are introduced. 
   2. Identify L4-staged animals by locating the white half-moon crescent shape of the developing vulva (Figure 5E).
      NOTE: Animals at the L4 stage are uniform in size and in body pigmentation. Pick animals with the white crescent to a fresh growth plate for examination of staged animals. The following day (~ 24 hours later) should be counted as adult day 1.
   3. Score a population of animals daily on adult day 2.
      NOTE: Exophers are typically scored at day 2 of adulthood, which is the peak exopher production under basal conditions. However, because the exophergenesis peak and timing may be shifted by environmental or genetic changes that are being studied, it is advised to score a population of adult animals daily over four days to generate the most comprehensive picture (Figure 3D).

5. Detection of exophers using a fluorescent microscope

1. Observe exophers using a high-magnification pseudo-stereo dissecting microscope that is outfitted for fluorescence microscopy.
2. Immobilize animals on NGM plates by pipetting 100-200 µL of 10-100 mM levamisole/tetramisole solution onto the NGM agar plate surface. After 2-4 minutes, the animals become paralyzed and can be observed directly on the agar plate.
   NOTE: Immobilization treatments are not absolutely required, such that with a trained eye, neuronal identification and exopher presence can be scored by visually following crawling animals under the microscope on the plate when determining whether or not an exopher has been produced.
3. Observe fluorescent neurons using a total magnification of 100x to accomplish dissecting microscope detection of exophers.
   NOTE: Scoring exopher events using dissection microscopy allows for the observation of large numbers of animals with relative ease directly on the agar plates on which they are reared.
4. Live imaging and mounting reporter strains for exopher studies using confocal microscopy
   1. Use a confocal microscope to live-image intracellular dynamics and characteristics of exophergenesis.
      NOTE: Live imaging is an advantageous approach for observing subtle details of exopher production because exopher production is a dynamic process.
   2. Restrict animal movement for high resolution live imaging using convenient methods, including utilization of levamisole or tetramisole at 10-100 mM or the application of hydrogel polystyrene microbeads (with diameters of 15 µm, 30 µm or 40 µm)¹⁸.
5. Slide preparation for compound and confocal microscopy
   1. Mount 20-50 animals into an immobilizing agent on a microscope slide. Reusable ringed cytology slides painted with 13 mm diameter raised-rings are useful for mounting.
   2. Pick live animals into 5-20 µL of a paralytic such as 10-100 mM levamisole or tetramisole within the painted circle or on the agar pad.
   3. Wait 4 minutes for paralysis, and then cover the slide with a coverslip (recommend No. 1½ (0.16 – 0.19 mm) or No. 2 (0.17 - 0.25 mm).
6. Mounting small numbers of animals
   1. Do not crush mounted animals; when observing only a few (less than 20) animals per slide, there is a risk of crushing some of the animals due to unequal pressure of the coverslip. This risk can be minimized by using a low percentage agarose pad for mounting.
   2. Make a 2-4% agarose pad slide, and then add 2-15 µL of paralytic solution to the pad. Keep in mind levamisole and tetramisole diffuse into the pad, decreasing their effective concentration.
   3. Mount by picking animals into a 2-15 µL drop of paralytic solution or microbeads resting on the agar pad. Place the coverslip on top and check that the animals are intact¹⁸.
7. Agar pad preparation
   1. To prepare 2% agar pads, heat 2% agarose in M9 solution and microwave until the agarose is in a homogeneous and molten state.
   2. To achieve an agar pad of sufficient quality, alternate mixing and microwaving at low power for less than 20 seconds. Avoid the inclusion of air bubbles within the pad by placing boiling agar on a heating block and allowing the bubbles to rise to the surface.
   3. Use a Pasteur pipette to draw agar from deep within the molten solution below the risen bubbles.
   4. Prepare two taped slides and place on either side of a clean glass microscope slide upon a flat surface. To make the taped slides place two 5 cm strips of laboratory tape upon each slide (Figure 6A).
   5. Using a Pasteur pipet, place a single drop of agar upon the clean microscope slide sandwiched between the taped slides (Figure 6B).
   6. Carefully and quickly, cover the drop of molten agar with a fourth clean slide by placing in across the taped slides (Figure 6Cc).
      NOTE: The slide should gently press the molten agar into a flattened circle about 0.4 mm thick (the thickness of the tape) (Figure 6D). The agar should quickly cool.
   7. Remove the top slide by sliding it off (Figure 6E). Agar pads dry quickly and are best used within minutes. Once the top slide is removed, use the gel pad immediately for mounting animals. Avoid using pads with air bubbles.
   8. Store agar pads up to 30 minutes encased between the two glass slides. Dried agar causes animals to clump together and desiccate. Mount animals within 2-15 µL of paralytic solution or microbeads and cover with coverslip; screen the slide within 20 minutes of paralysis and mounting (Figure 6).
      NOTE: Because stress conditions can alter exopher rates, avoid paralytics that can induce oxidative stress (ex: sodium azide) when screening for exophers.
8. Detection of exophers using a spinning-disk confocal microscope
   1. Observe cell biological features such as organelles and other contents with 1.4 numerical aperture objectives at 63x and 100x.
   2. Use software capable of stage-control and image acquisition utilizing the multidimensional acquisition. Microscopes and image processing software should also be suitable for imaging and data collection as these steps involve standard imaging approaches. 

6. Identifying touch neurons and scoring for exophers with mounted animals

1. Mount paralyzed adult animal (Figure 6).
2. Identify the desired Z-plane. Use low magnification bright-field (10-40x) to identify the suitable Z-plane of the animal, taking note of the positioning of the animal, head-tail orientation, and location of the vulva - which are landmarks for later neuronal and exopher identification (Figure 3A & Figure 5E).
3. Focus on the fluorescence signal of the chosen reporter. Staying in the same Z-plane, switch to widefield fluorescence viewing at 10-40x for the chosen cytosolic reporter.
   NOTE: In this example fluorescent expression is driven by the mec-4 mechanosensory touch neuron-specific promoter. High copy arrays, and different fluorophores have variability in expression and therefore variable fluorescent intensity. Adjust if needed.
4. Scroll within the Z axis to observe the depth of the animal and fluorescent expression in the focal plane. While doing so, confirm the head-tail orientation; the head/pharynx will have the fluorescent nerve ring and in this case, the tail will contain 1-2 visible PLM somas (Figure 3A).
5. Identify touch neurons 
   1. Identify whether the animal is mounted on the left or the right side (Figure 3A).
      NOTE: Considering the 3-dimensionality of the animal, the best imaging resolution is accomplished on the side closest to the optics.
   2. Identify the soma (ALM, ALMR, AVM) by observing - start at the head to identify the nerve ring and lateral neuronal processes.
   3. At 10-40x magnification, slowly scroll through the Z-axis to identify the attached process.
   4. Once the process is identified, follow it laterally in the posterior direction towards the vulva, where the soma will be apparent, marked by a round cell body at the end of the process. Once the most in-focus neuronal soma is found, it can be identified by using other neuronal landmarks as follows:
   5. Use the AVM, a nearby ventral neuron, to help assign animal orientation. If the AVM neuron is in the same plane as the ALM then the animal is resting upon its side and the neuron outside that plane is the ALMR . If the AVM neuron is not in the same plane as the ALM in question, the closest touch neuron to the focal plane is ALML.
   6. Identify the PVM neuron, another ventral touch neuron located near the tail, to indicate whether the anterior touch neuron is in the same plane. If so, the touch neuron observed is ALML. 
   7. Get a sense of the position of other soma bodies, near the area of interest (fluorescent neurons located on either side of the soma), and in all Z-planes, even if it is not possible to get the deepest neuron set in clear focus.
      NOTE: The identification of all touch neuron somas is important because out-of-focus soma can be mistaken for exophers. 

7. Identifying and scoring for exophers

1. Once a touch neuron is found, inspect it for large protrusions (exopher domains) large enough to be considered a bud exopher, (reaching at least 1/5^th the size of the originating soma) (Figure 1C).
   NOTE: The average exopher measures around 2-8 µm in diameter, while the average soma of a (ZB4065 bzIs166[Pmec-4::mCherry]) animal measures 6-10 µm in day 2 adults (Figure 7B).
2. If no bud or exopher domain is observed, inspect the neuronal soma for an attached thin filament emanating from the soma. Attached exophers tend to be located closer to the originating soma and in a similar Z-plane. 
   NOTE: Exophers do not always remain attached to the soma. The detection of an attached filament is a definitive indication that the object is an exopher.
3. To identify an unattached exopher, look for the contents of an exopher. Exophers can concentrate expelled fluorescent proteins and are therefore often brighter than the soma.
   NOTE: The contents of exophers are heterogeneous and variable. Cellular organelles such as lysosomes and mitochondria can also be extruded within exophers (Figure 4C-E).
4. Look for unattached exophers in different focal planes than the plane in which the originating soma was found. Although exophers have been seen to protrude from the ALM soma in any direction, it is typical that exophers protrude away from the soma, in a posterior direction from the neuronal process. 
5. Check for large, spherical objects that are not positioned and identified as neuronal somas. Exophers can be irregularly shaped but are typically spherical structures. Exophers become degraded over time, so older exophers tend to have a more irregular shape.
   NOTE: Mature or older exophers are distinguished from the dispersed "starry night" stage via the brighter fluorescence intensity of exophers and their spherical shape.
6. Investigate the “starry night” phenotype as evidence of earlier exophergenesis. Exophers progress into a “starry night” stage as the exopher breaks up into smaller vesicles and the surrounding hypodermis attempts to degrade the exopher contents (Figure 1G, 2B, 3B & 7A).
   NOTE: The starry night stage is marked by fragmented and scattered (sometimes networked) fluorescent entities that have lost structural integrity and displays a dim fluorescence in comparison to the touch neurons and exopher structures.
7. Look for instances of “multiple exopher events”. Exophers are usually produced as a singular occurrence (1 exopher emanating from 1 soma) but under some circumstances more than one exopher can be released from a single soma (Figure 7D).
   NOTE: Mature exophers can degrade into multiple vesicles as they are degraded in the hypodermis. Distinguishing whether each exopher was generated by an independent exophergenesis event or whether one original exopher split to create an additional vesicle can only be determined by time-lapse observation.
8. Keep in mind that not all morphological abnormalities mature into exophers.
   1. Do not score a distended soma as an exopher. An extended or pointed soma can be observed on occasion (especially with age or under stress), but an extension without a clear constriction site is not scored as an exopher.
   2. Reject small resolved buds that do not attain 1/5^th the size of the soma in exopher event quantification.
   3. Do not count neurite outgrowths as exophers. Mature neurites can extend dramatically with age (usually in the opposite direction of the neuronal process) and fluorescent protein can migrate into the distal end of such structures¹⁹.
      NOTE: These neurite outgrowths are not exophers as they have a distinct developmental pattern over days and weeks, do not form buds, and do not detach (Figure 7E).
9. Identify fluorescent entities that are not exophers.
   NOTE: It is important to get an idea of background fluorescence to ensure correct identification of the extruded fluorescent entity vs. autofluorescence.
   1. Transgenic fluorescent expression vs autofluorescence. Do not mistake autofluorescence for transgenic expression. The true exopher signal will not be in the intestine or gut (DIC confirmation can be used to identify these tissues) and exopher signal will be significantly brighter than background autofluorescence. 
      NOTE: Autofluorescence is caused by gut granule intestinal fluorescent pigmentation and accumulates with age. It is heterogenous, especially as viewed with different wavelengths.
   2. Signal from embryos. Do not mistake embryo signal for exophergenesis. Confirm suspicions of embryo signal by switching from fluorescence to brightfield illumination and checking for associations of signal with eggs in the uterus.
   3. Out of plane or nearby soma bodies. Avoid mistaking an out of plane soma for an exopher by identifying all nearby soma bodies, even out-of-focus somas at the start of the observation.
      NOTE: If scoring for exophers from ALMR, identify and account for the location of AVM and ALMR somas. More details on soma body identification are described in Figure 3A.

8. Scoring and statistics

1. Score exophers as binary (yes, there is an exopher/no, there is not an exopher).
2. Consider exopher detection as an "exopher-event" for a given neuron. An exopher event can constitute observation of a single exopher near a soma or multiple exophers.
   NOTE: To quantify numbers of individual exophergenesis events use time-lapse observation.
3. Count exopher events per a particular identified cell because different cells do not produce exophers at the same rate (see for example Figure 3C). ALMR neurons produce the most baseline exophers in the strains described herein and thus often this is the cell selected for exopher quantification from touch receptor neurons.
4. For statistics, in general, conduct at least 3 biological trials, of at least 30 animals scored per trial with the corresponding number of observations required for analysis of the disruption.
   1. For multiple trials involving one or two mutants/treatments compared to control, the Cochran-Mantel-Haenszel test is appropriate to determine p values.
   2. For trials involving more than two mutants of treatments compared to control, it is also appropriate to use a binary logistic regression analysis to evaluate significance for any number of categorical predictors.

Discussion

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 “starry night’ 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.

Materials

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