The toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease1,2. Accordingly, neurons invest considerable cellular resources in chaperones, protein degradation, autophagy and mitophagy to maintain proteostasis and mitochondrial quality3,4. Complicating the challenges of neuroprotection, misfolded human disease proteins and mitochondria can move into neighbouring cells via unknown mechanisms, which may promote pathological spread5,6. Here we show that adult neurons from Caenorhabditis elegans extrude large (approximately 4 μm) membrane-surrounded vesicles called exophers that can contain protein aggregates and organelles. Inhibition of chaperone expression, autophagy or the proteasome, in addition to compromising mitochondrial quality, enhances the production of exophers. Proteotoxically stressed neurons that generate exophers subsequently function better than similarly stressed neurons that did not produce exophers. The extruded exopher transits through surrounding tissue in which some contents appear degraded, but some non-degradable materials can subsequently be found in more remote cells, suggesting secondary release. Our observations suggest that exopher-genesis is a potential response to rid cells of neurotoxic components when proteostasis and organelle function are challenged. We propose that exophers are components of a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, which, when dysfunctional or diminished with age, might actively contribute to pathogenesis in human neurodegenerative disease and brain ageing.
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We thank B. Grant for expert advice, C. Reina for time-lapse microscopy help; N. Kane and J. Kramer for confocal microscopy assistance; and H. Ushakov for construction of some genetic lines. We thank G. Perumal and F. Macaluso for help with HPF fixations, and C. Crocker for the cartoon in Extended Data Fig. 2b. F. Sesti and M. Hilliard supplied C. elegans strains; A. Mendenhall, B. Sands and R. Brent constructed the MOSCI mitoTimer strain. Research was supported by the National Institutes of Health under award numbers 1R01NS086064 and 1R01AG046358. I.M. and R.J.G. were supported by the National Institute of General Medical Sciences under award number T32 GM008339. K.C.N. and D.H.H. were supported by NIH OD10943 (to D.H.H.); Core EM facilities (D.H.H.) NICHD P30 HD71593 for the RFK-IDDRC at Albert Einstein College of Medicine. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
The authors declare no competing financial interests.
Reviewer Information Nature thanks C. Bargmann, D. Divizio and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, An exopher is generated with evident filling and growth. The exopher size increased for more than 1 h, specifically in the exopher compartment, possibly via continual delivery of materials to the exopher after initial formation (see Supplementary Video 2). Strain is Is[Pmec-4mCh1], adult day 2. b, An ALMR soma with multiple exophers. Strain is Is[Pmec-4mCh2], adult day 2. c, A rare instance of an ALM neuron with exophers that appear extruded from the dendrite (arrows). Strain is Is[Pmec-4mCh1], adult day 2. d, Size measurements for somas (squares) and exophers (circles). Data are combined for exophers scored in different backgrounds, n = 35. Values analysed in Extended Data Table 2. e, Example of a exopher derived from a dye-filled amphid neuron. We identified exophers from dye-filled amphids, some of which appeared attached. Neuronal exophers are thus not induced solely in response to expression of foreign proteins, but can be produced from neurons that express only native proteins. See Extended Data Fig. 6c for a second example of a dye-filled exopher. f, Early adult longitudinal time-course on DiI-soaked wild-type N2. Dye-filled chemosensory amphid neurons also produce exophers with a peak early in life in wild-type animals. The production of exophers in this study reflects the extrusion of native neuronal contents, as no fluorescent transgene is introduced. Total n > 150, 3 trials. g, h, Dopaminergic PDE and CEP neurons can produce exophers. g, GFP-expressing PDE neuron with an anterior exopher indicated (8 out of 450 had exophers, typical of the low rate observed with GFP reporters in touch neurons). h, CEP neuron with an associated exopher. Strain is egIs1[Pdat-1GFP]; adult day 2. i, ASER neurons can form exophers. Strain is sesIs25[Pflp-6Aβ; Pgcy-5GFP]32; adult day 2. j, The onset of touch neuron exophers in an hsf-1 mutant background occurs 1 day earlier than wild-type touch neurons, beginning on adult day 1, but follows the general trend of high incidence early in adult life. Longitudinal study with Is[Pmec-4GFP]; hsf-1(sy441) (2 green trials, starting n = 25), and Is[Pmec-4mCh1]; hsf-1(sy441) (red, starting n = 25). We observed a similar temporal pattern for Is[Pmec-4mCh2]; [Pmec-3Q128CFP] (data not shown). A late onset peak might not be apparent owing to sickness of hsf-1 mutants later in life. Data in d and f are mean ± s.e.m. Data in j are from single longitudinal trials and thus error bars are not included. Scale bars, 2 μm.
a, A membrane GFP reporter in a PVM neuron reveals that exophers contain membrane. Strain is Pmec-4PH(plcDelta)::GFP (ref. 35). b, Relationship of exopher to ALMR soma. Schematic view from a lateral aspect (anterior to the left). ALMR soma remains connected to its primary dendrite. Several smaller membrane-bound tubes extend away from the soma, containing small expelled items, such as the large vesicle shown. The ALMR nucleus (N) is intact, but pushed to an eccentric position by cytoplasmic inclusions. The soma contains intact rough endoplasmic reticulum (RER), mitochondria (M), small vesicles (not shown), and larger protein aggregates (A). The exopher comprises heterogeneous contents. Exopher exterior is completely bounded by hypodermal plasma membrane, so exopher contents are not in immediate contact with hypodermal cytoplasm. A double membrane is often observed where the exopher is likely to supply the inner bilayer, and the hypodermis contributes the outer bilayer. Additional, separate membrane-bound items lie peripherally (not shown; but see h), which may be breakdown products from an earlier, larger stage of the exopher. Most internal exopher contents have their own membrane boundaries, but diffuse material (not shown; but see c, e) fills spaces around those membrane-bound objects. Membrane-bound contents include portions of neuronal cytoplasm holding intact RER, large protein aggregates, and complex whorls of membrane (W) that seem to enclose empty space. Two lysosomes (L) are shown, one in the process of fusing to exopher outer membrane. A tube is shown extending (far right) towards the pseudocoelom, which might offer a route for elimination of contents that cannot be degraded during hypodermal transit. Cartoon designed by and published with permission of C. Crocker. c, d, f, h, TEM views of an exopher, emitted from cell body in e. c, The exopher is fully embedded within hypodermis (H), seen from anterior aspect (thus left/right reversed). Strain is Is[Pmec-4mCh1]; Is[Pmec-4GFP]. C, animal cuticle. Exopher is 1.5 μm, similar in size to the excretory canal (EC), and lies closer to the pseudocoelom (P), whereas the ALMR neuron soma lies closer to cuticle (see e). Jagged white spaces running vertically through hypodermis are an artefact where tissue cracked during processing. d, The exopher is characterized by many small round protrusions and involuted portions, with multiple membrane layers. The main exopher complex has a complete plasma membrane surrounding it. e, The originating ALMR neuron still has intact cell and nuclear membranes. Aggregate within soma is not membrane bound, resembling a mammalian aggresome42. Electron density of the neuronal cytoplasm is darker than that of surrounding hypodermis, and mitochondria of hypodermis are far larger than those of the neuron. f, The exopher is surrounded by a continuous membrane and contains electron-lucent materials and electron-dense membrane whorls. g, Fluorescent microscopy of exopher in a day 2 animal, expressing soluble YFP, CFP-tagged Q128 fusion, and aggregation-prone mCherry. mCherry has a bright spot that excludes the other two signals (arrowhead). Q128–CFP and YFP are localized in the middle and bottom of exopher, respectively. YFP signal also forms a dim ring around mCherry spot. Lateral aspect shown, as in a. h, Thin sections (1–7, from 50 serial sections) through an exopher reveal a complex and heterogeneous structure. Additional membrane-bound objects at fringe of exopher main body (see panels 6, 7) may represent portions that have decayed from the original larger object, and are perhaps more easily phagocytosed by hypodermis, or shuttled along a tube for release into pseudocoelom. A small electron dense lysosome (L) lies beside exopher in panel 5. Scale bars, 2 μm (a, c, e, g), 1 μm (d, f, h).
Extended Data Figure 3 Fluorescence recovery after photo-bleaching (FRAP) and post-axotomy calcium imaging indicate that exophers that appear connected to the soma can fill with cytoplasmic materials.
a, Both connected and unconnected exophers can be identified at high frequency in the same strain with a 40× objective. Strain is Is[Pmec-4mCh1], adult day 2; n = 77 total, 3 trials. Data are mean ± s.e.m. b, Example of a connected ALMR exopher recovering after laser bleaching. Strain is Is[Pmec-4mCh1]; Is[Pmec-4GFP], adult day 2. Before laser treatment is ‘0 s’, other times are post-treatment. c, Example of a detached ALMR exopher photo-bleaching and failing to refill. Strain is Is[Pmec-4mCh1]; Is[Pmec-4GFP], adult day 2. d, Fluorescence recovery measurements reveal that some connected exophers are able to transport fluorescent material from the cytoplasm to the exopher, whereas disconnected exophers do not appear to transport fluorescent material. Shown are data for examples in b (red, connected) and c (blue, unconnected) above. e, Time-lapse measurements of fluorescence intensity of the soma (blue trace) and the exopher (red trace) during the laser axotomy experiment in f show that the injury-induced calcium wave in the soma is followed by a pulse of calcium increase in the exopher (red arrow, laser axotomy at t = 0 s). We analysed two individual neurons with exophers connected to the soma and three individual neurons with what appeared to be non-connected exophers. Only the clearly connected exophers gave a calcium response comparable (~100% signal) to that measured at the cell soma as in this panel. Strain is ZB4059 bzIs163[Pmec-4::GCaMP3.0::SL2::mCherry]. f, The soma calcium wave induced by laser axotomy is followed by a calcium wave to connected exophers. We laser-cut an ALMR neuron that had a connected exopher in a day 2 adult that expressed both mCherry and the calcium-sensitive fluorophore, GCaMP3. We made the laser cut 20 μm along the axon (yellow arrow) at time t = 0, while taking simultaneous time-lapse images (1 frame per 5 s). Selected frames are shown at t = −20 s (before laser axotomy), right after laser axotomy at t = 5 s (note increased fluorescence in the soma; white arrow), and at t = 15 s and 25 s post-axotomy (note the later exopher increase in fluorescence; white arrowheads). Signal quantification is in e. Supplementary Video 3 shows the calcium wave that travels from soma to exopher.
GFP-tagged lysosomes in touch neurons (bzIs168[Pmec-7LMP-1::GFP]) can be extruded in exophers in two types of lysosomal arrangements: (1) those that have small lysosome-like concentrated fluorescence, with mCherry dispersed (d), and (2) those that are nearly fully loaded with lysosome-like staining in which mCherry is also present throughout. a, Neuron soma featuring typical two large LMP-1::GFP-tagged pericentric lysosome domains, with no smaller ones evident. Strain is ZB4509 Is[Pmec-4mCh1]; bzIs168[Pmec-7LMP-1::GFP], green channel shown. As observed previously43, the LMP-1::GFP signal clearly marks the plasma membrane (M), but less intensely than the lysosomes (L). b, LMP-1::GFP reveals lysosome inclusions are frequent and sometimes prominent in exophers. Strain is ZB4070 bzIs168[Pmec-7LMP-1::GFP]. We found that 18 out of 25 (72%) of exophers scored contained lysosomes in day 2 adults. Note that LMP-1::GFP faintly labels membrane43 and rings the exopher in b–d, supporting that the exopher is membrane-bound. c, d, Co-labelling of aggregating mCherry and lysosome compartments suggests two types of lysosomal organization in exophers. Strain is ZB4509 Is[Pmec-4mCh1]; bzIs168[Pmec-7LMP-1::GFP]. c, Some exophers appear to be filled with LMP-1::GFP and coincident mCherry. d, LMP-1::GFP-tagged lysosomes included in exophers can be small and differentially localized from mCherry. In the absence of stress, neurons typically feature two large intensely fluorescent pericentric lysosome domains with LMP-1::GFP, with few smaller ones evident (see a). Neurons that had an exopher tended to also have additional small mobile lysosomes that we did not observe in cells without an exopher (see b–d). Additionally, neurons that featured ‘large lysosome’ exophers generally appeared to have fewer of the large perinuclear lysosomes in the soma (example in d). Scale bars, 2 μm. The inclusion of lysosomes in exophers suggests that some elimination of expelled material might be accomplished via internal degradation. Alternatively, dysfunctional lysosomes might be expelled via exophers.
Extended Data Figure 5 Mitochondria GFP markers exhibit a normal mitochondrial appearance in exophers.
a, Mitochondria in exophers can form a network. Strain is Is[Pmec-4mitoLS::ROGFP]; Is[Pmec-4mCh1]). Shown is an exopher budding off from the ALMR soma. The exopher contains a disproportionate number of punctate mCherry aggregates, and also includes a GFP signal typical in size for neuronal mitochondria. The mitochondria in the exopher exhibit a filamentous structure similar to those in the soma, and the signal does not co-localize with the mCherry signal but may remain within a distinct sub-cellular domain. These two observations are consistent with the mitoGFP label localized to actual mitochondria as opposed to representing mislocalized GFP-labelled protein. b, Exophers can contain punctate mitochondria, networked mitochondria, or no mitochondria. Strain is Is[Pmec-4mitoLS::ROGFP]; Is[Pmec-4mCh1]). In the left exopher, the mitoROGFP signal is localized to two puncta. The middle exopher contains networked mitochondria. The right exopher contains no visible mitoROGFP signal. c, Zoomed out view of b, to show location of exophers relative to the touch neuron soma. Scale bars, 2 μm (a–c). d, MitoTimer fluorescent reporter reveals a difference in the mitochondrial matrix oxidation environment in exophers versus somas. We used a single-copy Pmec-4MitoTimer reporter to measure the relative red/green signal in exopher–soma pairs. Exophers have proportionately more oxidized signal, suggesting ‘older’ mitochondria (with more oxidation of the matrix-localized reporter) are preferentially expelled. n = 7 exophers, single exopher mean ± s.e.m. *P < 0.05, paired t-test. e, Pharmacological disruption of mitochondria leads to higher rates of mitochondrial inclusion in exophers. Strain Is[Pmec-4mCh1]; zhsEx17[Pmec-4mitoLS::ROGFP] was treated with 230 μM juglone, which increases ROS production. Exophers from ALMR neurons exposed to juglone (blue, n = 30 total exophers) were significantly more likely to include at least one mitochondrion than untreated control exophers (white, n = 22 total exophers); 3 trials. Mitochondrial extrusion increases under conditions of juglone-induced oxidative stress. Data are mean ± s.e.m. **P < 0.01, unpaired t-test.
Extended Data Figure 6 RNAi knockdown of ced-1 and ced-6, but not other engulfment machinery, increases occurrence of mutiple exopher detection.
a, RNAi knockdown of ced-1 and ced-6 engulfment genes increases the number of ALMR neurons with ≥2 exophers near the touch neurons, supporting conclusions from mutants. Control is empty vector, strain is ZB4071 bzIs169[Pmec-18sid-1Psng-1YFP]; bzIs101[Pmec-4mCherry], at least 3 trials each, n > 30 ALMRs measured per trial; n > 15 cells with exopher per condition graphed. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, one-way ANOVA with Dunnett’s test. ced-1 and ced-6 RNAi do not increase the percentage of ALMRs that produce exophers (data not shown). b, Phosphatidylserine indicator annexinV::GFP (ref. 44) labels apoptotic corpses, but not mCherry-labelled exophers in strain ZB4083 smIS76[Phsp-16ANV::GFP]; Is[Pmec-4mCh1]. Phosphatidylserine can be recognized on corpses of necrotic touch neurons, showing that touch neurons can produce surface phosphatidylserine and be recognized by annexinV-tagging, when inappropriately induced to die33,44. 0 out of 18 fluorescent mCherry-labelled exophers were co-labelled with annexinV::GFP. ced-1 RNAi in the annexinV::GFP line did not improve phosphatidylserine detection on exophers (n = 0 out of 25 additional observations; not shown). c, In a DiI-soaked N2 animal, an amphid exopher originating from the ASIR soma can be seen proximal to the terminal bulb of the pharynx (P). The anterior coelomocytes ccAR and ccPR (C) also contain DiI, which must have been taken in and then jettisoned by the chemosensory amphid neurons and subsequently engulfed, analogously to the mCherry detected in Is[Pmec-4mCh1] coelomocytes. Thus, coelomocytes can scavenge the contents of exophers that are generated under normal physiological conditions, without the added stress of a potentially aggregating product of a transgene. Scale bar, 5 μm. d, In ZB4082 cup-4(ok837); Is[Pmec-4mCh1] mutants in which coelomocyte uptake is disrupted, an increased incidence of dispersed (D) fluorescence occurs (bracket denotes ‘starry night phenotype’, present in 29 out 200 animals, adult day 4). Similar dispersions are rare in cup-4(+) lines. Scale bar, 2 μm. AVM soma is also visible. e, Young adult animals that produced an exopher often later exhibit the starry night phenotype, suggesting that mCherry material can move through the body. Is[Pmec-4mCh1] animals were separated into populations that had an ALMR exopher on adult days 2 or 3 (blue arrows), and a population that had no ALMR exopher on days 2 or 3 (white arrows). Animals were scored again on day 5 for presence of the starry night phenotype. In the exopher-producing and non-exopher-producing groups, 42% and 6%, respectively, of animals exhibited a starry night phenotype. n = 60 total per group, 3 trials. Data are mean ± s.e.m. *P < 0.05, unpaired t-test. Arrow thickness is weighted according to relative incidence. Note differences are likely to be underestimated here, as the ‘no exopher’ category should include animals that have produced exophers, but were not present at the time of sampling.
As neurotoxic events such as protein aggregation or mitochondrial dysfunction occur in the cell, several homeostatic mechanisms clear them (left panel). At the young adult transition point to adult proteostasis (heat shock response down, unfolded protein response down, proteasome activity up12,13,14) or when basal levels of damage reach a threshold and overwhelm neuronal proteostasis, aggregates and organelles such as mitochondria and lysosomes are sequestered into a compartment that can be jettisoned from the cell. This compartment might include aggresomes described in mammalian cells42. For touch neurons, extruded exopher contents may be degraded by accompanying lysosomes, digested by the surrounding hypodermis, or may be re-extruded and reach the pseudocoelom to be taken up by coelomocytes. The process of exopher-genesis appears to be neuroprotective in young adults, but when dysregulated, might induce toxicity in neighbouring tissues. We speculate that exopher contents that cannot be degraded or passed on could remain in the neighbouring cell, where they could contribute to dysfunction. Exopher-genesis may be akin to the process by which protein aggregates and mitochondria become localized to neighbouring cells in humans, promoting the spread of disease.
Strain is Is[pmec-4mCh2]. ALM neuron with mCherry-visualized cytoplasm and aggregates. (MP4 355 kb)
S indicates the soma of an ALM neuron on adult day 2 with mCherry visualized; E indicates the significant extrusion of a balloon-like exopher, which grows with time. We noted that the size of this exopher increased for more than an hour, with fluorescence intensity increasing specifically in the exopher compartment, possibly via continual delivery of materials to the exopher after the initial formation. Strain is Is[pmec-4mCh1]. (MP4 98 kb)
We laser-cut an ALMR neuron that had a connected exopher in a day 2 adult that expressed both mCherry(bottom) and the calcium sensitive fluorophore, GCaMP3(top). Video shows the calcium wave that travels from soma to exopher. (MP4 236 kb)
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Melentijevic, I., Toth, M., Arnold, M. et al. C. elegans neurons jettison protein aggregates and mitochondria under neurotoxic stress. Nature 542, 367–371 (2017). https://doi.org/10.1038/nature21362
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