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

We characterize the in vivo mechanisms of aggregate and organelle extrusion from neurons via a process called exophergenesis, poorly-understood biology that's likely relevant to neurodegenerative disease pathology spread. These approaches are necessary for achieving reproducible scoring of extruded neuronal exophers and demonstrate a platform for the mechanistic dissection of cellular trash elimination via neuronal transfer to neighboring cells. Defining the in vivo mechanisms of how neurons eject aggregates may actually influence the design of novel strategies to aid in the treatment of human neurodegenerative conditions.

For basal conditions, house C.Elegans of the same biological age at a constant temperature of 20 degrees Celsius. For live imaging of the intracellular dynamics and characteristics of exophergenesis, place up to 20 animals onto an agaros pad on a glass microscope slide, and immobilize the animals. Carefully place a cover slip over the paralyzed worms and place the slide onto the stage of a confocal fluorescence microscope.

Use a 10 to 40x objective to identify the desired Z plane in Brightfield, taking notes of the positioning of the worm, the head tail orientation, and the vulva location, which are landmarks for later neuronal and exopher identification. Staying in the same Z plane, switch to widefield fluorescence viewing at 10 to 40x for the chosen cytosolic reporter, and scroll within the Z axis to observe the depth of the animal and the fluorescence expression in the focal plane. The head will have a fluorescent nerve ring, and the more pointed tail will contain one to two visible posterior lateral touch neuron somas.

Higher magnification with a 63x lens can help to identify the nerve ring, neuronal processes, and soma bodies. It is first helpful to identify the nerve ring located in the head of the animal and lateral neuronal processes. To do so, start at the head of the worm and slowly scroll through the Z axis to identify the plane of the nerve ring where the neuronal processes are attached.

Once the nerve ring has been identified, in fluorescence view follow the attached neuronal process laterally in the posterior direction toward the vulva, where the soma will be apparent. Once the round soma body or bodies are in focus, it is important to identify all the neurons nearby. For touch neuron identification, first find the ALML, ALMR, and AVM soma, which should be the brightest signals and marked by a round cell body at the end of the process.

Once the most in-focus neuronal soma has been located, use the AVM cell, a ventral cell, to help assign the orientation. If the AVM neuron is in the same plane as the ALM, then the animal is resting on its side and the lateral neuron 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 the ALML neuron.

Also helpful is to identify the PVM neuron, which is a ventral touch neuron located near the tail. The focus plane of the PVM will indicate whether the anterior touch neuron is the ALML or ALMR. If the ALM in question is in the same plane as the PVM, then the observed touch neuron is the ALML neuron.

After the most in-focus ALM has been identified and assigned, it's important to determine the positions of the other soma bodies near the area of interest and in all of the Z planes, as to not mistake them for exophers. Once the touch neuron of interest has been located, the ALMR or ALML, inspect the neuron for large protruding exopher domains, large enough to be considered a bud exopher, which is at least one fifth the size of the originating soma. If no bud or exopher domain is observed, further inspect the neuronal soma for an attached thin filament emanating from the soma body.

Attached exophers tend to be located closer to the originating soma and in a similar Z plane. To identify an unattached exopher, look for concentrated expelled fluorescent proteins, which are often brighter than the soma. Although depicted here, there is one large exopher.

There may be more than one fluorescent entity originating from a single soma. Look for unattached exophers in different focal planes and in the distant lateral region from where the originating soma was found. The exophers typically protrude away from the soma in a posterior direction from the neuronal process.

Check for large spherical objects that are not positioned and identified as neuronal somas. Although exophers are typically sphyrical structures, they can become degraded over time, acquiring a more irregular shape. Search for the presence of starry night events and for instances of multiple exopher events.

To avoid mistaking and out-of-plane soma for an exopher, it is critical to identify all nearby soma bodies, even out-of-focus somas at the start of the observation. An extended or pointed soma can be observed, but an extension without a clear constriction site should not be scored as an exopher. Reject small resolved buds that do not attain one fifth the size of the soma in exopher event quantification.

Although mature neurites can extend dramatically with age, and fluorescent proteins can migrate into the distal end of such structures, do not count neurite outgrowths as exophers. To identify fluorescent entities that are not exophers, be sure to exclude any autofluorescence, which may be mistaken for starry night vesicular debris under widefield fluorescence. To exclude embryonic fluorescent signals, switch between fluorescence and brightfield illumination, and check for association of the signal with eggs within the uterus.

This table provides a summary of different touch neuron expressed fluorescent reporters that have been used to monitor exopher production. Cargoes that are known to be extruded in exophers include aggregates such as Q128, a fusion of human Huntington expanded polyglutamine repeats, lysosomes GFP tagged with lysosomal associated membrane protein, and mitochondria tagged with matrix-localized GFP. Cytoplasmic GFP is not strongly expelled, and is preferentially retained in the soma, although GFP can be used to weakly visualize exophers.

In general, exopher production by the ALMR neuron-expressing mCherry under basal conditions begins on day one of adulthood and ranges from about five to 25%of ALMRs examined within the main exopher production timeframe of adult day one to three. Particular stresses and genetic perturbations can greatly increase exopher production within ALMR neurons. It is crucial to locate all of the nearby neurons before inspecting the neuron of interest and the surrounding area for exopher domains, intact exophers, or starry night events.

When you are comfortable with manually identifying exophers, enhanced throughput methods for large-scale screening using high-content imaging can be employed, allowing whole genome analysis and mechanistic exophergenesis dissection.