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IFD-1 and IFD-2 colocalize to juxtanuclear inclusions that become larger with age and are distinct from other organelles Solid white lines outline the touch neuron soma. Scale bar = 2 µm. a Ad2 touch neuron, DIC-GFP merge, strain expressing bzIs166[Pmec-4mCherry]; ifd-1(ok2404); bzSi3[Pmec-7GFP::IFD-1]. Image is representative of N > 40 neurons. b Ad2 touch neuron, DIC-GFP merge, strain expressing bzIs166[Pmec-4mCherry]; bzSi37[Pmec-7mNeonGreen::IFD-2]. Image is representative of N > 20 neurons with visible mNG expression for this strain. c Top: L4 larval stage ALM (Anterior Lateral Microtubule) neuron Bottom: Ad11 ALM neuron. Strain expresses bzIs166[Pmec-4mCherry]; bzSi3[Pmec-7GFP::IFD-1]. Image is representative of N > 50 neurons. d Adult touch neuron expressing bzEx279[Pmec-7GFP::IFD-1 Pmec-7RFP::IFD-2]. Image is representative of N > 20 neurons. e Adult touch neuron expressing bzIs166[Pmec-4mCherry]; bzEx265[Pmec-4TagBFP::AMAN-2]; bzSi3[Pmec-7GFP::IFD-1]. Image is representative of N > 20 neurons. f Adult touch neuron expressing bzIs3[Pmec-7GFP::IFD-1]; pwSi222[Pmec-7LMP-1::mScarlet]. Image is representative of N > 20 neurons. g Adult touch neuron expressing bzIs166[Pmec-4mCherry]; bzEx265[Pmec-4TagBFP::AMAN-2]; bzSi3[Pmec-7GFP::IFD-1]. Image is representative of N > 20 neurons. h Colocalization correlation of GFP::IFD-1 and RFP::IFD-2 signals (d) graphed as Pearson’s Coefficient of red and green channel; boxes indicate the coefficient range with a minimum (0.74), maximum (0.97), and mean (0.89); N = 10 neurons. i Colocalization correlation of GFP::IFD-1 and TagBFP::AMAN-2 signals (e) graphed as Pearson’s Coefficient of blue and green channel; boxes indicate the coefficient range, with a minimum (0.34), maximum (0.62), and mean (0.50); N = 10 neurons. j Colocalization correlation of GFP::IFD-1 and LMP-1::mSc signals (f) graphed as Pearson’s Coefficient of red and green channel; boxes indicate the coefficient range, with a minimum (0.05), maximum (0.47), and mean is (0.42); N = 10 neurons. k Colocalization correlation of RFP::IFD-2 and mitoROGFP (g) graphed as Pearson’s Coefficient of red and green channel; boxes indicate the coefficient range, with a minimum (0.11), maximum (0.39), and mean (0.26); N = 10 neurons.
Source publication
Toxic protein aggregates can spread among neurons to promote human neurodegenerative disease pathology. We found that in C. elegans touch neurons intermediate filament proteins IFD-1 and IFD-2 associate with aggresome-like organelles and are required cell-autonomously for efficient production of neuronal exophers, giant vesicles that can carry aggr...
Citations
... Study of aggregate transfer in the context of the mammalian brain is a major experimental challenge as events are rare, sporadic, and transiently apparent, and tissue is not easily accessible for in vivo observation. We model aggregate transfer by proteostressed ALMR touch receptor neurons in the living C. elegans nervous system (Melentijevic et al., 2017;Cooper et al., 2021;Arnold et al., 2023), an experimental system that enables molecular and genetic manipulation and evaluation in a physiological context, directly through the transparent cuticle (Corsi et al., 2015). ...
... More specifically, C. elegans adult neurons can extrude large vesicles called exophers (~5 µm; 100 X larger than exosomes) that carry potentially deleterious proteins and organelles out of the neuron (Melentijevic et al., 2017;Cooper et al., 2021;Arnold et al., 2023). Disrupting proteostasis via diminished chaperone expression, autophagy, or proteasome activity, or over-expressing aggregating proteins like human Alzheimer's disease associated fragment Aβ 1-42 , expanded polyglutamine Q128 protein, or high concentration mCherry fluorophore, increases exopher production from the affected neurons. ...
... Bottom panels are representative pictures (n > 100, scale bar = 10 μm) of an ALMR neuron without (lower left) or with (lower right) exopher production from strain ZB4065 bzIs166[P mec-4 ::mCherry], which expresses elevated mCherry in the touch receptor neurons. Over-expression of mCherry in bzIs166 is associated with enlargement of lysosomes and formation of large mCherry foci that often correspond to LAMP::GFP-positive structures; ultrastructure studies reveal considerable organelle morphological change not seen in low reporter-expression neurons; polyQ74, polyQ128, Aβ 1-42 over-expression all increase exophers (Melentijevic et al., 2017;Arnold et al., 2023). Most genetic compromise of different proteostasis branches--heat shock chaperones, proteasome, and autophagy--enhance exophergenesis, supporting exophergenesis as a response to proteostress. ...
Large vesicle extrusion from neurons may contribute to spreading pathogenic protein aggregates and promoting inflammatory responses, two mechanisms leading to neurodegenerative disease. Factors that regulate the extrusion of large vesicles, such as exophers produced by proteostressed C. elegans touch neurons, are poorly understood. Here, we document that mechanical force can significantly potentiate exopher extrusion from proteostressed neurons. Exopher production from the C. elegans ALMR neuron peaks at adult day 2 or 3, coinciding with the C. elegans reproductive peak. Genetic disruption of C. elegans germline, sperm, oocytes, or egg/early embryo production can strongly suppress exopher extrusion from the ALMR neurons during the peak period. Conversely, restoring egg production at the late reproductive phase through mating with males or inducing egg retention via genetic interventions that block egg-laying can strongly increase ALMR exopher production. Overall, genetic interventions that promote ALMR exopher production are associated with expanded uterus lengths and genetic interventions that suppress ALMR exopher production are associated with shorter uterus lengths. In addition to the impact of fertilized eggs, ALMR exopher production can be enhanced by filling the uterus with oocytes, dead eggs, or even fluid, supporting that distention consequences, rather than the presence of fertilized eggs, constitute the exopher-inducing stimulus. We conclude that the mechanical force of uterine occupation potentiates exopher extrusion from proximal proteostressed maternal neurons. Our observations draw attention to the potential importance of mechanical signaling in extracellular vesicle production and in aggregate spreading mechanisms, making a case for enhanced attention to mechanobiology in neurodegenerative disease.
... Exophers contain organelles, large protein complexes, and other components, and they play a role in the clearance of damaged, degraded, or aggregated material as well as dysfunctional mitochondria. Studies have demonstrated that exophers play a crucial role in neurological pathologies, as evidenced by research conducted by Melentijevic et al. (2017) and Arnold et al. (2023) [33,34]. Limited evidence is available regarding the involvement of exophers in tumors. ...
Cancer poses a significant public health challenge worldwide, and timely screening has the potential to mitigate cancer progression and reduce mortality rates. Currently, early identification of most tumors relies on imaging techniques and tissue biopsies. However, the use of low-cost, highly sensitive, non-invasive detection methods for early cancer screening has become more attractive. Extracellular Vesicles (EVs) released by all living cells contain distinctive biological components, such as nucleic acids, proteins, and lipids. These vesicles play crucial roles in the tumor microenvironment and intercellular communication during tumor progression, rendering liquid biopsy a particularly suitable method for diagnosis. Nevertheless, challenges related to purification methods and validation of efficacy currently hinder its widespread clinical implementation. These limitations underscore the importance of refining isolation techniques and conducting comprehensive investigations on EVs. This study seeks to evaluate the potential of liquid biopsy utilizing blood-derived EVs as a practical, cost-effective, and secure approach for early cancer detection.
... Apoptosis is important for both developmental processes and homeostatic mechanisms in 33 metazoans. The apoptosis pathway involves Bcl-2 Homology (BH) 3-only proteins, which are 34 conserved and function by inhibiting BCL-2 proteins, releasing components necessary for 35 ...
... autonomously for exopher production [33] . Additionally, the autophagy protein ATG-16.2 has been 216 found to promote exopher production cell autonomously [34] . ...
While traditionally studied for their pro-apoptotic functions, recent research suggests BH3-only proteins also have non-apoptotic roles. Here, we find that EGL-1, the BH3-only protein in Caenorhabditis elegans , promotes the cell-autonomous production of exophers in adult neurons. Exophers are large, micron-scale vesicles that are ejected from the cell and contain cellular components such as mitochondria. EGL-1 facilitates exopher production potentially through regulation of mitochondrial dynamics. Moreover, an endogenous, low level of EGL-1 expression appears to benefit dendritic health. Our findings provide insights into the mechanistic role of BH3-only protein in mitochondrial dynamics, downstream exopher production, and ultimately neuronal health.
Significance statement
BH3-only proteins were known for their function in inducing cell death. Their presence in healthy adult neurons, however, suggests additional roles. Our study focused on the BH3-only protein EGL-1 in the nematode Caenorhabditis elegans , where its apoptotic role was discovered. We reveal a new role in cell-autonomously promoting exopher production – a process where neurons extrude large vesicles containing potentially harmful cell contents. EGL-1 appears to promote this by regulating mitochondrial dynamics. We also report that low levels of EGL-1 benefit neuronal health and function. These findings expand our understanding of BH3-only proteins, mitochondrial dynamics, and exopher production in neurons and provide insights for neurodegenerative diseases.
... Exophers represent a novel type of extracellular vesicle (EV) recently discovered in Caenorhabditis elegans neurons 18 and body wall muscles 19 . Exophers extrude from juxtanuclear plasma membrane areas 20 as long nanotubules which can reach up to 4 µm in diameter at the distal end 21 . In C. elegans, exopherformation depends on cellular stressors 17,24 and mechanistically involves the delivery of cargo to aggresome-like organelles 20 . ...
... Exophers extrude from juxtanuclear plasma membrane areas 20 as long nanotubules which can reach up to 4 µm in diameter at the distal end 21 . In C. elegans, exopherformation depends on cellular stressors 17,24 and mechanistically involves the delivery of cargo to aggresome-like organelles 20 . Exopher-like processes have morphologically been reported in mammalian systems [22][23][24][25][26] and are thought to represent a protective strategy to remove proteotoxic protein aggregates 21, 27 and dysfunctional organelles such as mitochondria 22,24 . ...
... The copyright holder for this preprint this version posted April 7, 2024. ; https://doi.org/10.1101/2024.04.04.588146 doi: bioRxiv preprint neuronal exopher-formation 20 . In podocytes, 14-3-3 is a known synaptopodin interacting protein 49 , playing a role in the downstream function of cyclosporin A 49 . ...
Background
Membranous nephropathy (MN) is caused by autoantibody binding to podocyte foot process antigens such as THSD7A and PLA 2 R1. The mechanisms of the glomerular antigen/autoantibody deposition and clearance are unknown.
Methods
We explore the origin and significance of glomerular accumulations in (1) diagnostic and follow-up biospecimens from THSD7A ⁺ and PLA 2 R1 ⁺ -MN patients compared to nephrotic non-MN patients, and (2) in experimental models of THSD7A ⁺ -MN.
Results
We discovered podocyte exophers as correlates of histological antigen/autoantibody aggregates found in the glomerular urinary space of MN patients. Exopher vesicle formation represents a novel form of toxic protein aggregate removal in Caenorhabditis elegans neurons. In MN patients, podocytes released exophers to the urine. Enrichment of exophers from MN patient urines established them as a glomerular exit route for antigens and bound autoantibody. Exophers also carried disease-associated proteins such as complement and provided a molecular imprint of podocyte injury pathways. In experimental THSD7A ⁺ -MN, exophers were formed from podocyte processes and cell body. Their formation involved the translocation of antigen/autoantibody from the subepithelial to the urinary side of podocyte plasma membranes. Urinary exopher-release correlated with lower albuminuria and lower glomerular antigen/autoantibody burden. In MN patients the prospective monitoring of urinary exopher abundance and of exopher-bound autoantibodies was additive in the assessment of immunologic MN activity.
Conclusions
Exopher-formation and release is a novel pathomechanism in MN to remove antigen/autoantibody aggregates from the podocyte. Tracking exopher-release will add a non-invasive diagnostic tool with prognostic potential to clinical diagnostics and follow-up of MN patients.
... Exophers represent a novel type of extracellular vesicle (EV) recently discovered in Caenorhabditis elegans neurons 18 and body wall muscles 19 . Exophers extrude from juxtanuclear plasma membrane areas 20 as long nanotubules which can reach up to 4 µm in diameter at the distal end 21 . In C. elegans, exopher-formation depends on cellular stressors 17,24 and mechanistically involves the delivery of cargo to aggresome-like organelles 20 . ...
... Exophers extrude from juxtanuclear plasma membrane areas 20 as long nanotubules which can reach up to 4 µm in diameter at the distal end 21 . In C. elegans, exopher-formation depends on cellular stressors 17,24 and mechanistically involves the delivery of cargo to aggresome-like organelles 20 . Exopher-like processes have morphologically been reported in mammalian systems [22][23][24][25][26] and are thought to represent a protective strategy to remove proteotoxic protein aggregates 21,27 and dysfunctional organelles such as mitochondria 22,24 . ...
... To this end, 14-3-3 represented the most discerning marker in huIgG4 + -EVs. This is of interest, as 14-3-3 protein was recently shown to be required for exopher-formation in C. elegans neurons 20 . In comparison to nephrotic non-MN patients, huIgG4 + -EVs isolated from MN-patient urines contained abundant podocyte proteins such as THSD7A, PLA 2 R1, Nephrin, Podocin, and a-Actinin-4 by immunoblot or by immuno uorescence (Fig. 1D and Fig. S1F) showing their podocyte origin. ...
Background
Membranous nephropathy (MN) is caused by autoantibody binding to podocyte foot process antigens such as THSD7A and PLA2R1. The mechanisms of the glomerular antigen/autoantibody deposition and clearance are unknown.
Methods
We explore the origin and significance of glomerular accumulations in (1) diagnostic and follow-up biospecimens from THSD7A⁺ and PLA2R1⁺-MN patients compared to nephrotic non-MN patients, and (2) in experimental models of THSD7A⁺-MN.
Results
We discovered podocyte exophers as correlates of histological antigen/autoantibody aggregates found in the glomerular urinary space of MN patients. Exopher vesicle formation represents a novel form of toxic protein aggregate removal in Caenorhabditis elegans neurons. In MN patients, podocytes released exophers to the urine. Enrichment of exophers from MN patient urines established them as a glomerular exit route for antigens and bound autoantibody. Exophers also carried disease-associated proteins such as complement and provided a molecular imprint of podocyte injury pathways. In experimental THSD7A⁺-MN, exophers were formed from podocyte processes and cell body. Their formation involved the translocation of antigen/autoantibody from the subepithelial to the urinary side of podocyte plasma membranes. Urinary exopher-release correlated with lower albuminuria and lower glomerular antigen/autoantibody burden. In MN patients the prospective monitoring of urinary exopher abundance and of exopher-bound autoantibodies was additive in the assessment of immunologic MN activity.
Conclusions
Exopher-formation and release is a novel pathomechanism in MN to remove antigen/autoantibody aggregates from the podocyte. Tracking exopher-release will add a non-invasive diagnostic tool with prognostic potential to clinical diagnostics and follow-up of MN patients.
While traditionally studied for their proapoptotic functions in activating the caspase, research suggests BH3-only proteins also have other roles such as mitochondrial dynamics regulation. Here, we find that EGL-1, the BH3-only protein in Caenorhabditis elegans , promotes the cell-autonomous production of exophers in adult neurons. Exophers are large, micron-scale vesicles that are ejected from the cell and contain cellular components such as mitochondria. EGL-1 facilitates exopher production potentially through regulation of mitochondrial dynamics. Moreover, an endogenous, low level of EGL-1 expression appears to benefit dendritic health. Our findings provide insights into the role of neuronal BH3-only protein in mitochondrial dynamics, downstream exopher production, and ultimately neuronal health.
Extracellular vesicles (EVs) encompass a diverse array of membrane-bound organelles released outside cells in response to developmental and physiological cell needs. EVs play important roles in remodeling the shape and content of differentiating cells and can rescue damaged cells from toxic or dysfunctional content. EVs can send signals and transfer metabolites between tissues and organisms to regulate development, respond to stress or tissue damage, or alter mating behaviors. While many EV functions have been uncovered by characterizing ex vivo EVs isolated from body fluids and cultured cells, research using the nematode Caenorhabditis elegans has provided insights into the in vivo functions, biogenesis, and uptake pathways. The C. elegans EV field has also developed methods to analyze endogenous EVs within the organismal context of development and adult physiology in free-living, behaving animals. In this review, we summarize major themes that have emerged for C. elegans EVs and their relevance to human health and disease. We also highlight the diversity of biogenesis mechanisms, locations, and functions of worm EVs and discuss open questions and unexplored topics tenable in C. elegans, given the nematode model is ideal for light and electron microscopy, genetic screens, genome engineering, and high-throughput omics.
Large vesicle extrusion from neurons may contribute to spreading pathogenic protein aggregates and promoting inflammatory responses, two mechanisms leading to neurodegenerative disease. Factors that regulate extrusion of large vesicles, such as exophers produced by proteostressed C. elegans touch neurons, are poorly understood. Here we document that mechanical force can significantly potentiate exopher extrusion from proteostressed neurons. Exopher production from the C. elegans ALMR neuron peaks at adult day 2 or 3, coinciding with the C. elegans reproductive peak. Genetic disruption of C. elegans germline, sperm, oocytes, or egg/early embryo production can strongly suppress exopher extrusion from the ALMR neurons during the peak period. Conversely, restoring egg production at the late reproductive phase through mating with males or inducing egg retention via genetic interventions that block egg-laying can strongly increase ALMR exopher production. Overall, genetic interventions that promote ALMR exopher production are associated with expanded uterus lengths and genetic interventions that suppress ALMR exopher production are associated with shorter uterus lengths. In addition to the impact of fertilized eggs, ALMR exopher production can be enhanced by filling the uterus with oocytes, dead eggs, or even fluid, supporting that distention consequences, rather than the presence of fertilized eggs, constitute the exopher-inducing stimulus. We conclude that the mechanical force of uterine occupation potentiates exopher extrusion from proximal proteostressed maternal neurons. Our observations draw attention to the potential importance of mechanical signaling in extracellular vesicle production and in aggregate spreading mechanisms, making a case for enhanced attention to mechanobiology in neurodegenerative disease.
While autophagy genes are required for lifespan of long-lived animals, their tissue-specific roles in aging remain unclear. Here, we inhibited autophagy genes in Caenorhabditis elegans neurons, and found that knockdown of early-acting autophagy genes, except atg-16.2, increased lifespan, and decreased neuronal PolyQ aggregates, independently of autophagosomal degradation. Neurons can secrete protein aggregates via vesicles called exophers. Inhibiting neuronal early-acting autophagy genes, except atg-16.2, increased exopher formation and exopher events extended lifespan, suggesting exophers promote organismal fitness. Lifespan extension, reduction in PolyQ aggregates and increase in exophers were absent in atg-16.2 null mutants, and restored by full-length ATG-16.2 expression in neurons, but not by ATG-16.2 lacking its WD40 domain, which mediates noncanonical functions in mammalian systems. We discovered a neuronal role for C. elegans ATG-16.2 and its WD40 domain in lifespan, proteostasis and exopher biogenesis. Our findings suggest noncanonical functions for select autophagy genes in both exopher formation and in aging.
Large vesicle extrusion from neurons may contribute to spreading pathogenic protein aggregates and promoting inflammatory responses, two mechanisms leading to neurodegenerative disease. Factors that regulate extrusion of large vesicles, such as exophers produced by proteostressed C. elegans touch neurons, are poorly understood. Here we document that mechanical force can significantly potentiate exopher extrusion from proteostressed neurons. Exopher production from the C. elegans ALMR neuron peaks at adult day 2 or 3, coinciding with the C. elegans reproductive peak. Genetic disruption of C. elegans germline, sperm, oocytes, or egg/early embryo production can strongly suppress exopher extrusion from the ALMR neurons during the peak period. Conversely, restoring egg production at the late reproductive phase through mating with males or inducing egg retention via genetic interventions that block egg-laying can strongly increase ALMR exopher production. Overall, genetic interventions that promote ALMR exopher production are associated with expanded uterus lengths and genetic interventions that suppress ALMR exopher production are associated with shorter uterus lengths. In addition to the impact of fertilized eggs, ALMR exopher production can be enhanced by filling the uterus with oocytes, dead eggs, or even fluid, supporting that distention consequences, rather than the presence of fertilized eggs, constitute the exopher-inducing stimulus. We conclude that the mechanical force of uterine occupation potentiates exopher extrusion from proximal proteostressed maternal neurons. Our observations draw attention to the potential importance of mechanical signaling in extracellular vesicle production and in aggregate spreading mechanisms, making a case for enhanced attention to mechanobiology in neurodegenerative disease.