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.

Impaired signaling for neuromuscular synaptic maintenance is a feature of Motor Neuron Disease.

A central event in the pathogenesis of motor neuron disease (MND) is the loss of neuromuscular junctions (NMJs), yet the mechanisms that lead to this event in MND remain to be fully elucidated. Maintenance of the NMJ relies upon neural agrin (n-agrin) which, when released from the nerve terminal, activates the postsynaptic Muscle Specific Kinase (MuSK) signaling complex to stabilize clusters of acetylcholine receptors. Here, we report that muscle from MND patients has an increased proportion of slow fibers and muscle fibers with smaller diameter. Muscle cells cultured from MND biopsies failed to form large clusters of acetylcholine receptors in response to either non-MND human motor axons or n-agrin. Furthermore, levels of expression of MuSK, and MuSK-complex components: LRP4, Caveolin-3, and Dok7 differed between muscle cells cultured from MND patients compared to those from non-MND controls. To our knowledge, this is the first time a fault in the n-agrin-LRP4-MuSK signaling pathway has been identified in muscle from MND patients. Our results highlight the n-agrin-LRP4-MuSK signaling pathway as a potential therapeutic target to prolong muscle function in MND.

Neuron-epidermal attachment protects hyper-fragile axons from mechanical strain.

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/ß-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.

The metalloprotease ADM-4/ADAM17 promotes axonal repair.

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.

TDP-43 Mutation Affects Stress Granule Dynamics in Differentiated NSC-34 Motoneuron-Like Cells.

Amyotrophic Lateral Sclerosis (ALS) is characterized by degeneration of motor neurons in the brain and spinal cord. Cytoplasmic inclusions of TDP-43 are frequently reported in motor neurons of ALS patients. TDP-43 has also been shown to associate with stress granules (SGs), a complex of proteins and mRNAs formed in response to stress stimuli that temporarily sequester mRNA translation. The effect of pathogenic TDP-43 mutations within glycine-rich regions (where the majority of ALS-causing TDP-43 mutations occur) on SG dynamics in motor neurons is poorly understood. To address this issue, we generated murine NSC-34 cell lines that stably over-express wild type TDP-43 (TDP-43 W T ) or mutant forms (ALS-causing TDP-43 mutations TDP-43 A315T or TDP-43 M337V). We then differentiated these NSC-34 lines into motoneuron-like cells and evaluated SG formation and disassembly kinetics in response to oxidative or osmotic stress treatment. Wild type and mutant TDP-43 appeared to be largely retained in the nucleus following exposure to arsenite-induced oxidative stress. Upon arsenite removal, mutant TDP-43 clearly accumulated within HuR positive SGs in the cytoplasm, whereas TDP-43 W T remained mostly within the nucleus. 24 h following arsenite removal, all SGs were disassembled in both wild type and mutant TDP-43 expressing cells. By contrast, we observed significant differences in the dynamics of mutant TDP-43 association with SGs in response to hyperosmotic stress. Specifically, in response to sorbitol treatment, TDP-43 W T remained in the nucleus, whereas mutant TDP-43 relocalized to HuR positive SGs in the cytoplasm following exposure to sorbitol stress, resulting in a significant increase in TDP-43 SG numbers. These SGs remained assembled for 24 h following removal of sorbitol. Our data reveal that under certain stress conditions the rates of SG formation and disassembly is modulated by TDP-43 mutations associated with ALS, and suggest that this may be an early event in the seeding of insoluble cytoplasmic inclusions observed in ALS.

Modular transient nanoclustering of activated ß2-adrenergic receptors revealed by single-molecule tracking of conformation-specific nanobodies.

None of the current superresolution microscopy techniques can reliably image the changes in endogenous protein nanoclustering dynamics associated with specific conformations in live cells. Single-domain nanobodies have been invaluable tools to isolate defined conformational states of proteins, and we reasoned that expressing these nanobodies coupled to single-molecule imaging-amenable tags could allow superresolution analysis of endogenous proteins in discrete conformational states. Here, we used anti-GFP nanobodies tagged with photoconvertible mEos expressed as intrabodies, as a proof-of-concept to perform single-particle tracking on a range of GFP proteins expressed in live cells, neurons, and small organisms. We next expressed highly specialized nanobodies that target conformation-specific endogenous ß2-adrenoreceptor (ß2-AR) in neurosecretory cells, unveiling real-time mobility behaviors of activated and inactivated endogenous conformers during agonist treatment in living cells. We showed that activated ß2-AR (Nb80) is highly immobile and organized in nanoclusters. The Gαs-GPCR complex detected with Nb37 displayed higher mobility with surprisingly similar nanoclustering dynamics to that of Nb80. Activated conformers are highly sensitive to dynamin inhibition, suggesting selective targeting for endocytosis. Inactivated ß2-AR (Nb60) molecules are also largely immobile but relatively less sensitive to endocytic blockade. Expression of single-domain nanobodies therefore provides a unique opportunity to capture highly transient changes in the dynamic nanoscale organization of endogenous proteins.

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.

Epidermal control of axonal attachment via ß-spectrin and the GTPase-activating protein TBC-10 prevents axonal degeneration.

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/ß-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/ß-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 ß-spectrin functions in this context non-cell-autonomously. We propose a model in which mechanically resistant epidermal attachment structures are maintained by UNC-70/ß-spectrin and TBC-10 during movement, preventing axonal detachment and degeneration.

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.

Neuronal sub-compartmentalization: a strategy to optimize neuronal function.

Neurons are highly polarized cells that consist of three main structural and functional domains: a cell body or soma, an axon, and dendrites. These domains contain smaller compartments with essential roles for proper neuronal function, such as the axonal presynaptic boutons and the dendritic postsynaptic spines. The structure and function of these compartments have now been characterized in great detail. Intriguingly, however, in the last decade additional levels of compartmentalization within the axon and the dendrites have been identified, revealing that these structures are much more complex than previously thought. Herein we examine several types of structural and functional sub-compartmentalization found in neurons of both vertebrates and invertebrates. For example, in mammalian neurons the axonal initial segment functions as a sub-compartment to initiate the action potential, to select molecules passing into the axon, and to maintain neuronal polarization. Moreover, work in Drosophila melanogaster has shown that two distinct axonal guidance receptors are precisely clustered in adjacent segments of the commissural axons both in vivo and in vitro, suggesting a cell-intrinsic mechanism underlying the compartmentalized receptor localization. In Caenorhabditis elegans, a subset of interneurons exhibits calcium dynamics that are localized to specific sections of the axon and control the gait of navigation, demonstrating a regulatory role of compartmentalized neuronal activity in behaviour. These findings have led to a number of new questions, which are important for our understanding of neuronal development and function. How are these sub-compartments established and maintained? What molecular machinery and cellular events are involved? What is their functional significance for the neuron? Here, we reflect on these and other key questions that remain to be addressed in this expanding field of biology.

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.

Defects in synaptic transmission at the neuromuscular junction precede motor deficits in a TDP-43Q331K transgenic mouse model of amyotrophic lateral sclerosis.

Transactive response DNA-binding protein-43 (TDP-43) is involved in gene regulation via the control of RNA transcription, splicing, and transport. TDP-43 is a major protein component of ubiquinated inclusions that are found in amyotrophic lateral sclerosis (ALS); however, the function of TDP-43 at the neuromuscular junction (NMJ) and its role in ALS pathogenesis is largely unknown. Here, we show that TDP-43Q331K mutation in mice resulted in impaired neurotransmission by age 3 mo, preceding deficits in motor function and motor neuron loss, which were observed from age 10 mo. These defects were in the effective fusion and release of synaptic vesicles within the motor nerve terminal and manifested in decreased quantal content and reduced probability of quantal release. We observed morphologic alterations that were associated with the TDP-43Q331K mutation, such as aberrant innervation patterns and the distribution of synaptic vesicle-related proteins, which is indicative of a failing NMJ undergoing synaptic remodeling. These findings support a growing acceptance that dysregulation of the NMJ function is a key early event in the pathology of ALS.-Chand, K. K., Lee, K. M., Lee, J. D., Qiu, H., Willis, E. F., Lavidis, N. A., Hilliard, M. A., Noakes, P. G. Defects in synaptic transmission at the neuromuscular junction precede motor deficits in a TDP-43Q331K transgenic mouse model of amyotrophic lateral sclerosis.

6-OHDA-induced dopaminergic neurodegeneration in Caenorhabditis elegans is promoted by the engulfment pathway and inhibited by the transthyretin-related protein TTR-33.

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.

Phosphatidylserine save-me signals drive functional recovery of severed axons in Caenorhabditis elegans.

Functional regeneration after axonal injury requires transected axons to regrow and reestablish connection with their original target tissue. The spontaneous regenerative mechanism known as axonal fusion provides a highly efficient means of achieving targeted reconnection, as a regrowing axon is able to recognize and fuse with its own detached axon segment, thereby rapidly reestablishing the original axonal tract. Here, we use behavioral assays and fluorescent reporters to show that axonal fusion enables full recovery of function after axotomy of Caenorhabditis elegans mechanosensory neurons. Furthermore, we reveal that the phospholipid phosphatidylserine, which becomes exposed on the damaged axon to function as a "save-me" signal, defines the level of axonal fusion. We also show that successful axonal fusion correlates with the regrowth potential and branching of the proximal fragment and with the retraction length and degeneration of the separated segment. Finally, we identify discrete axonal domains that vary in their propensity to regrow through fusion and show that the level of axonal fusion can be genetically modulated. Taken together, our results reveal that axonal fusion restores full function to injured neurons, is dependent on exposure of phospholipid signals, and is achieved through the balance between regenerative potential and level of degeneration.

The Heterochronic Gene lin-14 Controls Axonal Degeneration in C. elegans Neurons.

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.

A multi-trap microfluidic chip enabling longitudinal studies of nerve regeneration in Caenorhabditis elegans.

Several sophisticated microfluidic devices have recently been proposed for femtosecond laser axotomy in the nematode C. elegans for immobilization of the animals for surgery to overcome time-consuming and labor-intensive manual processes. However, nerve regeneration studies require long-term recovery of the animals and multiple imaging sessions to observe the regeneration capabilities of their axons post-injury. Here we present a simple, multi-trap device, consisting of a single PDMS (polydimethylsiloxane) layer, which can immobilize up to 20 animals at the favorable orientation for optical access needed for precise laser surgery and high-resolution imaging. The new device, named "worm hospital" allows us to perform the entire nerve regeneration studies, including on-chip axotomy, post-surgery housing for recovery, and post-recovery imaging all on one microfluidic chip. Utilizing the worm hospital and analysis of mutants, we observed that most but not all neurodevelopmental genes in the Wnt/Frizzled pathway are important for regeneration of the two touch receptor neurons ALM and PLM. Using our new chip, we observed that the cwn-2 and cfz-2 mutations significantly reduced the reconnection possibilities of both neurons without any significant reduction in the regrowth lengths of the severed axons. We observed a similar regeneration phenotype with cwn-1 mutation in ALM neurons only.

Cortical synaptic and dendritic spine abnormalities in a presymptomatic TDP-43 model of amyotrophic lateral sclerosis.

Layer V pyramidal neurons (LVPNs) within the motor cortex integrate sensory cues and co-ordinate voluntary control of motor output. In amyotrophic lateral sclerosis (ALS) LVPNs and spinal motor neurons degenerate. The pathogenesis of neural degeneration is unknown in ALS; 10% of cases have a genetic cause, whereas 90% are sporadic, with most of the latter showing TDP-43 inclusions. Clinical and experimental evidence implicate excitotoxicity as a prime aetiological candidate. Using patch clamp and dye-filling techniques in brain slices, combined with high-resolution confocal microscopy, we report increased excitatory synaptic inputs and dendritic spine densities in early presymptomatic mice carrying a TDP-43Q331K mutation. These findings demonstrate substantive alterations in the motor cortex neural network, long before an overt degenerative phenotype has been reported. We conclude that increased excitatory neurotransmission is a common pathophysiology amongst differing genetic cases of ALS and may be of relevance to the 95% of sporadic ALS cases that exhibit TDP-43 inclusions.

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.

Neuron-specific knock-down of SMN1 causes neuron degeneration and death through an apoptotic mechanism.

Spinal muscular atrophy is a devastating disease that is characterized by degeneration and death of a specific subclass of motor neurons in the anterior horn of the spinal cord. Although the gene responsible, survival motor neuron 1 (SMN1), was identified 20 years ago, it has proven difficult to investigate its effects in vivo. Consequently, a number of key questions regarding the molecular and cellular functions of this molecule have remained unanswered. We developed a Caenorhabditis elegans model of smn-1 loss-of-function using a neuron-specific RNA interference strategy to knock-down smn-1 selectively in a subclass of motor neurons. The transgenic animals presented a cell-autonomous, age-dependent degeneration of motor neurons detected as locomotory defects and the disappearance of presynaptic and cytoplasmic fluorescent markers in targeted neurons. This degeneration led to neuronal death as revealed by positive reactivity to genetic and chemical cell-death markers. We show that genes of the classical apoptosis pathway are involved in the smn-1-mediated neuronal death, and that this phenotype can be rescued by the expression of human SMN1, indicating a functional conservation between the two orthologs. Finally, we determined that Plastin3/plst-1 genetically interacts with smn-1 to prevent degeneration, and that treatment with valproic acid is able to rescue the degenerative phenotype. These results provide novel insights into the cellular and molecular mechanisms that lead to the loss of motor neurons when SMN1 function is reduced.