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

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.

Figure 1:. Stages of Exophergenesis.

The process of making and ejecting an exopher is called ‘exopher-genesis’. The dynamic process of exopher formation can take several minutes to several hours. Depicted are examples of soma and exopher morphology at specific steps during the dynamic exophergenesis process in a high-exopher producing strain, ZB4065 bzIs166[P_mec-4mCherry]. All images are of day 2 adult ALM neurons taken with a 100x objective. (A) Normal soma. Adult mechanosensory touch neuron ALM transgenically expressing P_mec-4mCherry. The soma morphology depicted is typical of young adult neurons in this strain, with mCherry concentrations in the cytoplasm. (B) Early bud phase. The first observable step of exophergenesis involves polarization of selected cytoplasmic material to the edge of the soma membrane. This step is often accompanied by an expansion or swelling of the soma. In the case of the touch neurons, the pre-exopher domain (PED) extends into the surrounding hypodermis (not visible here). Note the greater concentration of mCherry material into the early bud domain. (C) Late bud phase. Upon further cellular polarization and an expansion of the pre-exopher domain, a constriction between the soma and exopher (arrow) becomes evident. This event signals the transition to the late bud phase. Although in the late bud stage the cell exhibits a clear constriction site and separate soma and exopher domains, it is not yet pinched off completely from the soma; the budding exopher may be attached by a thick stalk (arrow). The budding domain is considered an early exopher when the diameter of the exopher domain in question is roughly ⅓ larger than the diameter of the construction site/stalk. (D) Early-exopher phase. Early exophers can be attached by a stalk from the departing soma—the diameter of this connection can thin as the exopher moves away from the soma. Cytoplasmic material can be transferred from the soma to the exopher via this tube, although most material is loaded during the process of budding out. Exophers can detach from the soma as depicted in (E), separated exophers are considered mature exophers (F). The mature exopher can transit through the surrounding hypodermal tissue, moving away from the departing soma. (G) Breakdown of the mCherry-labelled exopher into smaller vesicles within the hypodermis results in a scattered punctate appearance of the mCherry material, most likely as it enters the hypodermal endolysosomal network. The dispersed punctate signal is called the “starry night” phase. Degradation of some exopher contents is likely accomplished by hypodermal lysosomes, but some material is not fully degraded and is often re-extruded by the hypodermis into the pseudocoelom. The post-exophergenesis mCherry transit is described in more detail in Figure 2.

Figure 2:. mCherry extruded from touch neurons in exophers engages the surrounding hypodermal lysosomal network but can later be extruded into the pseudocoelom where coelomocytes can store/degrade the mCherry.

(A) Cartoon summary of how mCherry extruded in exophers transits the body after expulsion by neurons. During exophergenesis selected cellular contents such as mCherry become localized and bud off from the sending neuronal soma in an independent vesicle surrounded by the neuronal and hypodermal plasma membranes. Since the touch neurons are embedded in the hypodermal tissue, as the exopher domain buds outwards it moves further into the hypodermis. The exopher can transit the hypodermis, and after hours to days, exopher contents can fragment within the endolysosomal network of the hypodermis. The mCherry can appear as scattered puncta throughout the hypodermis, a stage called “starry night”. After a few days, some of the mCherry can pass out of the hypodermis into the surrounding pseudocoelom, where scavenger cells called coelomocytes can get access to, and take up, mCherry that can be stored. (B) Example of the appearance of the starry night mCherry vesicles. Image of an ALM soma tagged with mCherry with large exopher fragments and starry night vesicles. Strain is ZB4065 bzIs166[P_mec-4mCherry]. (C) Example of mCherry concentration in distant coelomocytes. Sideview of an adult animal day 10 of strain ZB4065 bzIs166[P_mec-4mCherry] showing mCherry concentrated in coelomocytes (arrows). Some starry night vesicles are also evident. In general coelomocyte concentration becomes evident after about adult day 5 of life. (B bottom) Cartoon reproduction of (B), with touch neurons and processes outlined in red, as are brightest exopher fragments; scattered small vesicles of different Z-depths are shown in lighter pink. (C bottom) Cartoon version of image of (C), showing neuronal process in red, starry night in pink and coelomocytes in green.

Figure 3:. Mechanosensory touch neurons produce exophers at different levels with a precise temporal profile.

(A) (Top) Cartoon depiction of mechanosensory touch neurons in spatial relation to key anatomical landmarks of C. elegans including the pumping pharynx and the neuron-dense nerve ring at the head of the animal, the vulva at the mid body, and the tapered tail. (Bottom) Fluorescently labeled touch neurons expressing GFP as viewed from the top and left side (images adapted from WormAtlas). The red box depicts the area where ALM exophers are typically located. (B) High magnification view of the mid body region at which ALM-derived exophers are produced in a strain expressing [P_mec-4mCherry]. AVM and ALMR neuron are depicted, and shown is an ALMR exopher along with mCherry starry night. ALMR neurons most readily produce exophers. (C) ALMR mechanosensory touch neurons more readily produce exophers compared to other touch neurons in hermaphrodites under basal conditions. Mechanosensory touch neuron exopher production on adult day 2, as scored for individual touch receptor neurons is indicated. Strain: ZB4065 bzIs166[P_mec-4mCherry], N>150, error bars are SEM. (D) ALMR touch neurons produce more exophers during days 2 and 3 of adulthood compared with the adolescent L4 stage or with animals in advanced age. Strain: ZB4065 bzIs166[P_mec-4mCherry], N>150, error bars are SEM.

Figure 4:. Examples of some fluorescent reporters that tag exopher contents.

A straightforward way to observe exophers is by creating transgenic animals that express fluorophores from neuronal promoters. The fluorophores allow for visualization of the exopher and transgenic expression induces aggregation and/or proteostress that increases exophergenesis. Exophers produced by amphid neurons can also be observed under native conditions, using dye filling for visualization. Shown are examples of common strains that can be used to observe exophers, (E) exopher, (S) soma. (A) Soma and exopher from an ALM of an adult of strain SK4005 zdIs5[P_mec-4GFP], 100x objective used for photography, scale bar 3μm. In this strain, exophers that include soluble GFP are measured, but exopher production occurs infrequently. Fusing GFP to proteins that can be preferentially extruded in exophers in other studies confirms that GFP fusions can be detected in mature exophers. (B) ALM soma and exopher of an adult of strain ZB4065 bzIs166[P_mec-4mCherry], which expresses mCherry and induces touch neuron exopher production. 100x objective used for photography, scale bar 5 μm. (C) ALM soma and exopher of an adult of strain ZB4067 bzIs167[P_mec-4mitogfp P_mec-4mCherry4]; igIs1[P_mec-7YFP P_mec-3htt57Q128::cfp lin-15+]; selective blue channel used for image of htt57Q128::CFP. The exopher contains htt57Q128::CFP aggregates (arrows), that appear more concentrated in the exopher than in the soma. 40x objective used for photography, scale bar 5μm. (D-E) Exophers can contain organelles and organelle-specific tagging with fluorescent proteins enables monitoring of organelle extrusion. (D) Lysosomal membrane tag LMP-1::GFP outlines the soma and exopher membrane and tags plasma membranes weakly (plasma membrane localization is a trafficking step on the way to lysosomal targeting) and labels lysosomal organelles strongly. Shown is an adult ALM soma co-expressing P_mec-4mCherry and the P_mec-7LMP-1::GFP that localizes to membranes and lysosomes. The soma has an attached exopher with other smaller extrusions likely to be exopher fragments (arrows). GFP positive structures are included in the soma and are present in the large exopher, strain: ZB4509 bzIs166[P_mec-4mCherry]; bzIs168[P_mec-7LMP-1::GFP]. 100x objective used for photography, scale bar 5 μm. E) A mitochondrial GFP marker can be used to identify mitochondria in soma and exophers. Shown is an adult ALM soma expressing P_mec-4mCherry and mito::ROGFP, which localizes to the mitochondrial matrix. mito::ROGFP expressed alone, without the mCherry, can also readily be used to identify neurons and score for exophers that include mitochondria. Strain: ZB4528 bzIs166[P_mec-4mCherry]; zhsEx17 [P_mec-4mitoLS::ROGFP]. 100x objective used for photography; scale bar 5μm.

Figure 5:. Developmental cycle of C. elegans and L4 identification.

(A) At 20 °C an egg takes approximately 9 hours to hatch once laid by the mother. (B) A newly hatched animal is in larval stage 1 (L1) and molts into an L2 larva after 12 hours. (C) Animals remain in the L2 and the (D) L3 larval stages for about 8 hours each. (E) Adolescent animals are considered the fourth larval stage (L4) and are marked by a conspicuous developing vulva that appears as a white crescent near the mid body. The presence of this while crescent enables easy identification and picking of L4 staged animals to establish synchronized cultures that later facilitate scoring for exophers. Animals remain in the L4 stage for about 10 hours before their final molt into gravid adults, F) identified by developing eggs, visible spermatheca, and the initiation of egg-laying.

Figure 6:. Preparation of microscope slide agar pad.

(A) Prepare two slides with a single strip of laboratory tape placed lengthwise across the top. Place a non-taped microscope slide in between as pictured. B) Place a drop of molten agarose on top of the slide. (C) Place a clean slide gently on top of the drop, pressing the agarose into a deflated circle pad. (D) Remove the taped slides, which act to accomplish an even flattening of the agar that is needed to create an even pad. (E) Remove the top slide once the agarose pad has dried. (F) Pipette a paralytic solution (levamisole or tetramisole) on top of the agar pad. (G) Pick appropriately staged animals into the paralytic. (H) Gently cover the animals with a coverslip and ensure animals are alive.

Figure 7:. Characters of exophers and exopher identification criteria.

(A) General criteria that identify an exopher. (B) Diameter comparisons between the sending soma and the extruded exopher, measured in μm. Adult ALM somas, N=35, strain: ZB4065 bzIs166[P_mec-4mCherry] - 6.53 μm average size of soma and 3.83 μm average size of exopher. (C) Defining criteria for differentiating between an exopher domain and a budding exopher. (D) Most commonly, individual neurons make one large exopher, which later splits or fragments as the hypodermis attempts to degrade its contents. Still, multiple exophers may be observed next to one touch neuron that might derive from either multiple exopher events from one neuron or alternatively, exophers can also bud or fragment themselves. The origin of multiple exopher-like entities can only be determined using time lapse microscopy. Top depicts an ALMR touch neuron soma with a single distant exopher. Bottom depicts an ALMR touch neuron soma with multiple exopher-like extrusions. (E) Common morphological features in adult ALM touch neuron somas that may be mistaken for exopher events. Top left - A distended ALM soma, with no clear exopher domain or constriction site. Top middle - Neurons can have small extracellular protrusions that may be analogous to exophers, but do not meet size requirement criteria to be considered an exopher. Top right – With age, touch neurons can develop outgrowths along their minor neurite. Often mCherry material can be collected at the tip of the neurite outgrowth. This is not scored as an exopher if the collected mCherry does not meet exopher-to-soma size requirements. Bottom depicts adult ALM neurons that have defining criteria for an exopher domain or an exopher. Botom left - ALM soma that has a prominent exopher domain that selectively includes mCherry cytosol and mCherry tagged aggregates. The exopher domain constriction site is marked by arrows and meets the size criteria (at least 1/5^th the size of the soma). The largest diameter of the exopher domain is almost ⅓ bigger than the diameter of the constriction site, meeting criteria for an exopher event. Bottom middle - ALM soma that has a prominent budding exopher that meets the size criteria. There is a clear constriction site. Bottom right - ALM soma that has an attached mCherry-filled exopher that meets exopher size requirements. The exopher is attached by a thin connecting filament. All images are from strain ZB4065 bzIs166[P_mec-4mCherry].