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Axonal fusion is an efficient means of repair following axonal transection, whereby the regenerating axon fuses with its own separated axonal fragment to restore neuronal function. Despite being described over 50 years ago, its molecular mechanisms remain poorly understood. Here, we demonstrate that the Caenorhabditis elegans metalloprotease ADM-4, an ortholog of human ADAM17, is essential for axonal fusion. We reveal that animals lacking ADM-4 cannot repair their axons by fusion, and that ADM-4 has a cell-autonomous function within injured neurons, localizing at the tip of regrowing axon and fusion sites. We demonstrate that ADM-4 overexpression enhances fusion to levels higher than wild type, and that the metalloprotease and phosphatidylserine-binding domains are essential for its function. Last, we show that ADM-4 interacts with and stabilizes the fusogen EFF-1 to allow membranes to merge. Our results uncover a key role for ADM-4 in axonal fusion, exposing a molecular target for axonal repair.

Axons experience significant strain caused by organismal development and movement. A combination of intrinsic mechanical resistance and external shielding by surrounding tissues prevents axonal damage, although the precise mechanisms are unknown. Here, we reveal a neuroprotective function of neuron-epidermal attachment in Caenorhabditis elegans. We show that a gain-of-function mutation in the epidermal hemidesmosome component LET-805/myotactin, in combination with a loss-of-function mutation in UNC-70/β-spectrin, disrupts the uniform attachment and subsequent embedment of sensory axons within the epidermis during development. This generates regions of high tension within axons, leading to spontaneous axonal breaks and degeneration. Completely preventing attachment, by disrupting HIM-4/hemicentin or MEC-5/collagen, eliminates tension and alleviates damage. Finally, we demonstrate that progressive neuron-epidermal attachment via LET-805/myotactin is induced by the axon during development, as well as during regeneration after injury. Together, these results reveal that establishment of uniform neuron-epidermal attachment is critical to protect axons from mechanical strain during development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The 100-y-old neuron doctrine from Ramón y Cajal states that neurons are individual cells, rejecting the process of cell-cell fusion in the normal development and function of the nervous system. However, fusogens-specialized molecules essential and sufficient for the fusion of cells-are expressed in the nervous system of different species under conditions of viral infection, stress, or disease. Despite these findings, whether the expression of fusogens in neurons leads to cell-cell fusion, and, if so, whether this affects neuronal fate, function, and animal behavior, has not been explored. Here, using Caenorhabditis elegans chemosensory neurons as a model system, we provide proof-of-principle that aberrant expression of fusogens in neurons results in neuron-neuron fusion and behavioral impairments. We demonstrate that fusion between chemoattractive neurons does not affect the response to odorants, whereas fusion between chemoattractive and chemorepulsive neurons compromises chemosensation. Moreover, we provide evidence that fused neurons are viable and retain their original specific neuronal fate markers. Finally, analysis of calcium transients reveals that fused neurons become electrically coupled, thereby compromising neural circuit connectivity. Thus, we propose that aberrant expression of fusogens in the nervous system disrupts neuronal individuality, which, in turn, leads to a change in neural circuit connectivity and disruption of normal behavior. Our results expose a previously uncharacterized basis of circuit malfunction, and a possible underlying cause of neurological diseases., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Neurons are subjected to strain due to body movement and their location within organs and tissues. However, how they withstand these forces over the lifetime of an organism is still poorly understood. Here, focusing on touch receptor neuron-epidermis interactions using Caenorhabditis elegans as a model system, we show that UNC-70/β-spectrin and TBC-10, a conserved GTPase-activating protein, function non-cell-autonomously within the epidermis to dynamically maintain attachment of the axon. We reveal that, in response to strain, UNC-70/β-spectrin and TBC-10 stabilize trans-epidermal hemidesmosome attachment structures which otherwise become lost, causing axonal breakage and degeneration. Furthermore, we show that TBC-10 regulates axonal attachment and maintenance by inactivating RAB-35, and reveal functional conservation of these molecules with their vertebrate orthologs. Finally, we demonstrate that β-spectrin functions in this context non-cell-autonomously. We propose a model in which mechanically resistant epidermal attachment structures are maintained by UNC-70/β-spectrin and TBC-10 during movement, preventing axonal detachment and degeneration.

Following a transection injury to the axon, neurons from a number of species have the ability to undergo spontaneous repair via fusion of the two separated axonal fragments. In the nematode Caenorhabditis elegans , this highly efficient regenerative axonal fusion is mediated by epithelial fusion failure-1 (EFF-1), a fusogenic protein that functions at the membrane to merge the two axonal fragments. Identifying modulators of axonal fusion and EFF-1 is an important step toward a better understanding of this repair process. Here, we present evidence that the small GTPase RAB-5 acts to inhibit axonal fusion, a function achieved via endocytosis of EFF-1 within the injured neuron. Therefore, we find that perturbing RAB-5 activity is sufficient to restore axonal fusion in mutant animals with decreased axonal fusion capacity. This is accompanied by enhanced membranous localization of EFF-1 and the production of extracellular EFF-1-containing vesicles. These findings identify RAB-5 as a novel regulator of axonal fusion in C. elegans hermaphrodites and the first regulator of EFF-1 in neurons. SIGNIFICANCE STATEMENT Peripheral and central nerve injuries cause life-long disabilities due to the fact that repair rarely leads to reinnervation of the target tissue. In the nematode Caenorhabditis elegans , axonal regeneration can proceed through axonal fusion, whereby a regrowing axon reconnects and fuses with its own separated distal fragment, restoring the original axonal tract. We have characterized axonal fusion and established that the fusogen epithelial fusion failure-1 (EFF-1) is a key element for fusing the two separated axonal fragments back together. Here, we show that the small GTPase RAB-5 is a key cell-intrinsic regulator of the fusogen EFF-1 and can in turn regulate axonal fusion. Our findings expand the possibility for this process to be controlled and exploited to facilitate axonal repair in medical applications., (Copyright © 2019 the authors.)

Oxidative stress is linked to many pathological conditions including the loss of dopaminergic neurons in Parkinson's disease. The vast majority of disease cases appear to be caused by a combination of genetic mutations and environmental factors. We screened for genes protecting Caenorhabditis elegans dopaminergic neurons from oxidative stress induced by the neurotoxin 6-hydroxydopamine (6-OHDA) and identified the transthyretin-related gene ttr-33. The only described C. elegans transthyretin-related protein to date, TTR-52, has been shown to mediate corpse engulfment as well as axon repair. We demonstrate that TTR-52 and TTR-33 have distinct roles. TTR-33 is likely produced in the posterior arcade cells in the head of C. elegans larvae and is predicted to be a secreted protein. TTR-33 protects C. elegans from oxidative stress induced by paraquat or H2O2 at an organismal level. The increased oxidative stress sensitivity of ttr-33 mutants is alleviated by mutations affecting the KGB-1 MAPK kinase pathway, whereas it is enhanced by mutation of the JNK-1 MAPK kinase. Finally, we provide genetic evidence that the C. elegans cell corpse engulfment pathway is required for the degeneration of dopaminergic neurons after exposure to 6-OHDA. In summary, we describe a new neuroprotective mechanism and demonstrate that TTR-33 normally functions to protect dopaminergic neurons from oxidative stress-induced degeneration, potentially by acting as a secreted sensor or scavenger of oxidative stress.

The disproportionate length of an axon makes its structural and functional maintenance a major task for a neuron. The heterochronic gene lin-14 has previously been implicated in regulating the timing of key developmental events in the nematode C. elegans. Here, we report that LIN-14 is critical for maintaining neuronal integrity. Animals lacking lin-14 display axonal degeneration and guidance errors in both sensory and motor neurons. We demonstrate that LIN-14 functions both cell autonomously within the neuron and non-cell autonomously in the surrounding tissue, and we show that interaction between the axon and its surrounding tissue is essential for the preservation of axonal structure. Furthermore, we demonstrate that lin-14 expression is only required during a short period early in development in order to promote axonal maintenance throughout the animal's life. Our results identify a crucial role for LIN-14 in preventing axonal degeneration and in maintaining correct interaction between an axon and its surrounding tissue., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Axonal degeneration is a characteristic feature of neurodegenerative disease and nerve injury. Here, we characterize axonal degeneration in Caenorhabditis elegans neurons following laser-induced axotomy. We show that this process proceeds independently of the WLD(S) and Nmnat pathway and requires the axonal clearance machinery that includes the conserved transmembrane receptor CED-1/Draper, the adaptor protein CED-6, the guanine nucleotide exchange factor complex Crk/Mbc/dCed-12 (CED-2/CED-5/CED-12), and the small GTPase Rac1 (CED-10). We demonstrate that CED-1 and CED-6 function non-cell autonomously in the surrounding hypodermis, which we show acts as the engulfing tissue for the severed axon. Moreover, we establish a function in this process for CED-7, an ATP-binding cassette (ABC) transporter, and NRF-5, a lipid-binding protein, both associated with release of lipid-vesicles during apoptotic cell clearance. Thus, our results reveal the existence of a WLD(S)/Nmnat-independent axonal degeneration pathway, conservation of the axonal clearance machinery, and a function for CED-7 and NRF-5 in this process., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Functional regeneration after nervous system injury requires transected axons to reconnect with their original target tissue. Axonal fusion, a spontaneous regenerative mechanism identified in several species, provides an efficient means of achieving target reconnection as a regrowing axon is able to contact and fuse with its own separated axon fragment, thereby re-establishing the original axonal tract. Here we report a molecular characterization of this process in Caenorhabditis elegans, revealing dynamic changes in the subcellular localization of the EFF-1 fusogen after axotomy, and establishing phosphatidylserine (PS) and the PS receptor (PSR-1) as critical components for axonal fusion. PSR-1 functions cell-autonomously in the regrowing neuron and, instead of acting in its canonical signalling pathway, acts in a parallel phagocytic pathway that includes the transthyretin protein TTR-52, as well as CED-7, NRF-5 and CED-6 (refs 9, 10, 11, 12). We show that TTR-52 binds to PS exposed on the injured axon, and can restore fusion several hours after injury. We propose that PS functions as a 'save-me' signal for the distal fragment, allowing conserved apoptotic cell clearance molecules to function in re-establishing axonal integrity during regeneration of the nervous system.

Axonal degeneration arises as a consequence of neuronal injury and is a common hallmark of a number of neurodegenerative diseases. However, the genetic causes and the cellular mechanisms that trigger this process are still largely unknown. Based on forward genetic screening in C. elegans, we have identified the α-tubulin acetyltransferase gene mec-17 as causing spontaneous, adult-onset, and progressive axonal degeneration. Loss of MEC-17 leads to microtubule instability, a reduction in mitochondrial number, and disrupted axonal transport, with altered distribution of both mitochondria and synaptic components. Furthermore, mec-17-mediated axonal degeneration occurs independently from its acetyltransferase domain; is enhanced by mutation of coel-1, a tubulin-associated molecule; and correlates with the animal's body length. This study therefore identifies a critical role for the conserved microtubule-associated protein MEC-17 in preserving axon integrity and preventing axonal degeneration., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Microtubules have been known for decades to be basic elements of the cytoskeleton. They form long, dynamic, rope-like structures within the cell that are essential for mitosis, maintenance of cell shape, and intracellular transport. More recently, in vitro studies have implicated microtubules as signaling molecules that, through changes in their stability, have the potential to trigger growth of axons and dendrites in developing neurons. In this study, we show that specific mutations in the Caenorhabditis elegans mec-7/β-tubulin gene cause ectopic axon formation in mechanosensory neurons in vivo. In mec-7 mutants, the ALM mechanosensory neuron forms a long ectopic neurite that extends posteriorly, a phenotype that can be mimicked in wild-type worms with a microtubule-stabilizing drug (paclitaxel), and suppressed by mutations in unc-33/CRMP2 and the kinesin-related gene, vab-8. Our results also reveal that these ectopic neurites contain RAB-3, a marker for presynaptic loci, suggesting that they have axon-like properties. Interestingly, in contrast with the excessive axonal growth observed during development, mec-7 mutants are inhibited in axonal regrowth and remodeling following axonal injury. Together our results suggest that MEC-7/β-tubulin integrity is necessary for the correct number of neurites a neuron generates in vivo and for the capacity of an axon to regenerate.

Nervous system function requires proper development of two functional and morphological domains of neurons, axons and dendrites. Although both these domains are equally important for signal transmission, our understanding of dendrite development remains relatively poor. Here, we show that in C. elegans the Wnt ligand, LIN-44, and its Frizzled receptor, LIN-17, regulate dendrite development of the PQR oxygen sensory neuron. In lin-44 and lin-17 mutants, PQR dendrites fail to form, display stunted growth, or are misrouted. Manipulation of temporal and spatial expression of LIN-44, combined with cell-ablation experiments, indicates that this molecule is patterned during embryogenesis and acts as an attractive cue to define the site from which the dendrite emerges. Genetic interaction between lin-44 and lin-17 suggests that the LIN-44 signal is transmitted through the LIN-17 receptor, which acts cell autonomously in PQR. Furthermore, we provide evidence that LIN-17 interacts with another Wnt molecule, EGL-20, and functions in parallel to MIG-1/Frizzled in this process. Taken together, our results reveal a crucial role for Wnt and Frizzled molecules in regulating dendrite development in vivo., Competing Interests: The authors have declared that no competing interests exist.

A set of conserved molecules guides axons along the metazoan dorsal-ventral axis. Recently, Wnt glycoproteins have been shown to guide axons along the anterior-posterior (A/P) axis of the mammalian spinal cord. Here, we show that, in the nematode Caenorhabditis elegans, multiple Wnts and Frizzled receptors regulate the anterior migrations of neurons and growth cones. Three Wnts are expressed in the tail, and at least one of these, EGL-20, functions as a repellent. We show that the MIG-1 Frizzled receptor acts in the neurons and growth cones to promote their migrations and provide genetic evidence that the Frizzleds MIG-1 and MOM-5 mediate the repulsive effects of EGL-20. While these receptors mediate the effects of EGL-20, we find that the Frizzled receptor LIN-17 can antagonize MIG-1 signaling. Our results indicate that Wnts play a key role in A/P guidance in C. elegans and employ distinct mechanisms to regulate different migrations.

The alae, longitudinal ridges of the lateral cuticle, are the most visible specialization of the Caenorhabditis elegans surface. They are present only in L1 and dauer larvae and in adults. Little is known about the mechanisms through which at the appropriate stages secretion of cuticle components by the seam cells results in the formation of the alae. Here we show that three proteins, each containing a Zona Pellucida domain (ZP), are components of the cuticle necessary for larval alae development: CUT-1 and CUT-5 in dauer larvae and CUT-3 and CUT-5 in L1s. Transcriptional regulation of the corresponding genes contributes to the stage-specific role of these proteins. Larvae with reduced cut-1, cut-3 or cut-5 function not only lack alae but are also larger in diameter due to an increase in the width of the lateral cuticle. We propose a model in which reduction of the body diameter, which occurs in normal L1 and dauer larvae, is the result of a dorso-ventral shrinking of the internal layer of the lateral cuticle and formation of the alae results from the folding of the external layer of the lateral cuticle over the reduced, internal one. Alae of adults appear to form through a different mechanism.

An animal's ability to detect and avoid toxic compounds in the environment is crucial for survival. We show that the nematode Caenorhabditis elegans avoids many water-soluble substances that are toxic and that taste bitter to humans. We have used laser ablation and a genetic cell rescue strategy to identify sensory neurons involved in the avoidance of the bitter substance quinine, and found that ASH, a polymodal nociceptive neuron that senses many aversive stimuli, is the principal player in this response. Two G protein alpha subunits GPA-3 and ODR-3, expressed in ASH and in different, nonoverlapping sets of sensory neurons, are necessary for the response to quinine, although the effect of odr-3 can only be appreciated in the absence of gpa-3. We identified and cloned a new gene, qui-1, necessary for quinine and SDS avoidance. qui-1 codes for a novel protein with WD-40 domains and which is expressed in the avoidance sensory neurons ASH and ADL.