Fusogen-mediated neuron-neuron fusion disrupts neural circuit connectivity and alters animal behavior.

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.

EFF-1-mediated regenerative axonal fusion requires components of the apoptotic pathway.

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.

Disruption of RAB-5 Increases EFF-1 Fusogen Availability at the Cell Surface and Promotes the Regenerative Axonal Fusion Capacity of the Neuron.

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.

Cell-cell fusion in the nervous system: Alternative mechanisms of development, injury, and repair.

Over a century ago, the seminal work of Ramón y Cajal revealed that the nervous system is made of individual units, the neurons, which are related to each other by contiguity rather than continuity. This view overturned the idea that the nervous system was a reticulum of fibers, a rete diffusa nervosa, as proposed and defined by Camillo Golgi. Although the neuron theory has been widely confirmed in every model system studied and constitutes the basis of modern neuroscience, evidence accumulated over the years suggests that neurons, similar to other types of cells, have the potential to fuse their membranes and undergo cell-cell fusion under certain conditions. This concept adds a substantial layer to our view of the nervous system and how it functions. Here, we bring together past and more recent discoveries on multiple aspects of neuronal fusion, discussing how this cellular event is generated, and what consequences it has for our understanding of nervous system development, disease, injury, and repair.

Applying antibiotic selection markers for nematode genetics.

Antibiotic selection markers have been recently developed in the multicellular model organism Caenorhabditis elegans and other related nematode species, opening great opportunities in the field of nematode transgenesis. Here we describe how these antibiotic selection systems can be easily combined with many well-established genetic approaches to study gene function, improving time- and cost-effectiveness of the nematode genetic toolbox.

An antibiotic selection marker for nematode transgenesis.

We have developed a nematode transformation vector carrying the bacterial neomycin resistance gene (NeoR) and shown that it could confer resistance to G-418 on both wild-type Caenorhabditis elegans and C. briggsae. This selection system allows hands-off maintenance and enrichment of transgenic worms carrying non-integrated transgenes on selective plates. We also show that this marker can be used for Mos1-mediated single-copy insertion in wild-type genetic backgrounds (MosSCI-biotic).

The dynamin GTPase mediates regenerative axonal fusion in Caenorhabditis elegans by regulating fusogen levels.

Axonal fusion is a neuronal repair mechanism that results in the reconnection of severed axon fragments, leading to the restoration of cytoplasmic continuity and neuronal function. While synaptic vesicle recycling has been linked to axonal regeneration, its role in axonal fusion remains unknown. Dynamin proteins are large GTPases that hydrolyze lipid-binding membranes to carry out clathrin-mediated synaptic vesicle recycling. Here, we show that the Caenorhabditis elegans dynamin protein DYN-1 is a key component of the axonal fusion machinery. Animals carrying a temperature-sensitive allele of dyn-1(ky51) displayed wild-type levels of axonal fusion at the permissive temperature (15°C) but presented strongly reduced levels at the restrictive temperature (25°C). Furthermore, the average length of regrowth was significantly diminished in dyn-1(ky51) animals at the restrictive temperature. The expression of wild-type DYN-1 cell-autonomously into dyn-1(ky51) mutant animals rescued both the axonal fusion and regrowth defects. Furthermore, DYN-1 was not required prior to axonal injury, suggesting that it functions specifically after injury to control axonal fusion. Finally, using epistatic analyses and superresolution imaging, we demonstrate that DYN-1 regulates the levels of the fusogen protein EFF-1 post-injury to mediate axonal fusion. Together, these results establish DYN-1 as a novel regulator of axonal fusion.

SARS-CoV-2 infection and viral fusogens cause neuronal and glial fusion that compromises neuronal activity.

Numerous viruses use specialized surface molecules called fusogens to enter host cells. Many of these viruses, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can infect the brain and are associated with severe neurological symptoms through poorly understood mechanisms. We show that SARS-CoV-2 infection induces fusion between neurons and between neurons and glia in mouse and human brain organoids. We reveal that this is caused by the viral fusogen, as it is fully mimicked by the expression of the SARS-CoV-2 spike (S) protein or the unrelated fusogen p15 from the baboon orthoreovirus. We demonstrate that neuronal fusion is a progressive event, leads to the formation of multicellular syncytia, and causes the spread of large molecules and organelles. Last, using Ca2+ imaging, we show that fusion severely compromises neuronal activity. These results provide mechanistic insights into how SARS-CoV-2 and other viruses affect the nervous system, alter its function, and cause neuropathology.

Selectable genetic markers for nematode transgenesis.

The nematode Caenorhabditis elegans has been used to study genetics and development since the mid-1970s. Over the years, the arsenal of techniques employed in this field has grown steadily in parallel with the number of researchers using this model. Since the introduction of C. elegans transgenesis, nearly 20 years ago, this system has been extensively used in areas such as rescue experiments, gene expression studies, and protein localization. The completion of the C. elegans genome sequence paved the way for genome-wide studies requiring higher throughput and improved scalability than provided by traditional genetic markers. The development of antibiotic selection systems for nematode transgenesis addresses these requirements and opens the possibility to apply transgenesis to investigate biological functions in other nematode species for which no genetic markers had been developed to date.

Axonal fusion: An alternative and efficient mechanism of nerve repair.

Injuries to the nervous system can cause lifelong morbidity due to the disconnect that occurs between nerve cells and their cellular targets. Re-establishing these lost connections is the ultimate goal of endogenous regenerative mechanisms, as well as those induced by exogenous manipulations in a laboratory or clinical setting. Reconnection between severed neuronal fibers occurs spontaneously in some invertebrate species and can be induced in mammalian systems. This process, known as axonal fusion, represents a highly efficient means of repair after injury. Recent progress has greatly enhanced our understanding of the molecular control of axonal fusion, demonstrating that the machinery required for the engulfment of apoptotic cells is repurposed to mediate the reconnection between severed axon fragments, which are subsequently merged by fusogen proteins. Here, we review our current understanding of naturally occurring axonal fusion events, as well as those being ectopically produced with the aim of achieving better clinical outcomes.

The Apoptotic Engulfment Machinery Regulates Axonal Degeneration in C. elegans Neurons.

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.