A proton-inhibited DEG/ENaC ion channel maintains neuronal ionstasis and promotes neuronal survival under stress.

The nervous system participates in the initiation and modulation of systemic stress. Ionstasis is of utmost importance for neuronal function. Imbalance in neuronal sodium homeostasis is associated with pathologies of the nervous system. However, the effects of stress on neuronal Na+ homeostasis, excitability, and survival remain unclear. We report that the DEG/ENaC family member DEL-4 assembles into a proton-inactivated sodium channel. DEL-4 operates at the neuronal membrane and synapse to modulate Caenorhabditis elegans locomotion. Heat stress and starvation alter DEL-4 expression, which in turn alters the expression and activity of key stress-response transcription factors and triggers appropriate motor adaptations. Similar to heat stress and starvation, DEL-4 deficiency causes hyperpolarization of dopaminergic neurons and affects neurotransmission. Using humanized models of neurodegenerative diseases in C. elegans, we showed that DEL-4 promotes neuronal survival. Our findings provide insights into the molecular mechanisms by which sodium channels promote neuronal function and adaptation under stress.

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.

The diverse functions of the DEG/ENaC family: linking genetic and physiological insights.

The DEG/ENaC family of ion channels was defined based on the sequence similarity between degenerins (DEG) from the nematode Caenorhabditis elegans and subunits of the mammalian epithelial sodium channel (ENaC), and also includes a diverse array of non-voltage-gated cation channels from across animal phyla, including the mammalian acid-sensing ion channels (ASICs) and Drosophila pickpockets. ENaCs and ASICs have wide ranging medical importance; for example, ENaCs play an important role in respiratory and renal function, and ASICs in ischaemia and inflammatory pain, as well as being implicated in memory and learning. Electrophysiological approaches, both in vitro and in vivo, have played an essential role in establishing the physiological properties of this diverse family, identifying an array of modulators and implicating them in an extensive range of cellular functions, including mechanosensation, acid sensation and synaptic modulation. Likewise, genetic studies in both invertebrates and vertebrates have played an important role in linking our understanding of channel properties to function at the cellular and whole animal/behavioural level. Drawing together genetic and physiological evidence is essential to furthering our understanding of the precise cellular roles of DEG/ENaC channels, with the diversity among family members allowing comparative physiological studies to dissect the molecular basis of these diverse functions.

Physiological insight into the conserved properties of Caenorhabditis elegans acid-sensing degenerin/epithelial sodium channels.

Acid-sensing ion channels (ASICs) are members of the diverse family of degenerin/epithelial sodium channels (DEG/ENaCs). They perform a wide range of physiological roles in healthy organisms, including in gut function and synaptic transmission, but also play important roles in disease, as acidosis is a hallmark of painful inflammatory and ischaemic conditions. We performed a screen for acid sensitivity on all 30 subunits of the Caenorhabditis elegans DEG/ENaC family using two-electrode voltage clamp in Xenopus oocytes. We found two groups of acid-sensitive DEG/ENaCs characterised by being either inhibited or activated by increasing proton concentrations. Three of these acid-sensitive C. elegans DEG/ENaCs were activated by acidic pH, making them functionally similar to the vertebrate ASICs. We also identified three new members of the acid-inhibited DEG/ENaC group, giving a total of seven additional acid-sensitive channels. We observed sensitivity to the anti-hypertensive drug amiloride as well as modulation by the trace element zinc. Acid-sensitive DEG/ENaCs were found to be expressed in both neurons and non-neuronal tissue, highlighting the likely functional diversity of these channels. Our findings provide a framework to exploit the C. elegans channels as models to study the function of these acid-sensing channels in vivo, as well as to study them as potential targets for anti-helminthic drugs. KEY POINTS: Acidosis plays many roles in healthy physiology, including synaptic transmission and gut function, but is also a key feature of inflammatory pain, ischaemia and many other conditions. Cells monitor acidosis of their surroundings via pH-sensing channels, including the acid-sensing ion channels (ASICs). These are members of the degenerin/epithelial sodium channel (DEG/ENaC) family, along with, as the name suggests, vertebrate ENaCs and degenerins of the roundworm Caenorhabditis elegans. By screening all 30 C. elegans DEG/ENaCs for pH dependence, we describe, for the first time, three acid-activated members, as well as three additional acid-inhibited channels. We surveyed both groups for sensitivity to amiloride and zinc; like their mammalian counterparts, their currents can be blocked, enhanced or unaffected by these modulators. Likewise, they exhibit diverse ion selectivity. Our findings underline the diversity of acid-sensitive DEG/ENaCs across species and provide a comparative resource for better understanding the molecular basis of their function.

Distinct roles for two Caenorhabditis elegans acid-sensing ion channels in an ultradian clock.

Biological clocks are fundamental to an organism's health, controlling periodicity of behaviour and metabolism. Here, we identify two acid-sensing ion channels, with very different proton sensing properties, and describe their role in an ultradian clock, the defecation motor program (DMP) of the nematode Caenorhabditis elegans. An ACD-5-containing channel, on the apical membrane of the intestinal epithelium, is essential for maintenance of luminal acidity, and thus the rhythmic oscillations in lumen pH. In contrast, the second channel, composed of FLR-1, ACD-3 and/or DEL-5, located on the basolateral membrane, controls the intracellular Ca2+ wave and forms a core component of the master oscillator that controls the timing and rhythmicity of the DMP. flr-1 and acd-3/del-5 mutants show severe developmental and metabolic defects. We thus directly link the proton-sensing properties of these channels to their physiological roles in pH regulation and Ca2+ signalling, the generation of an ultradian oscillator, and its metabolic consequences.

Biological clocks regulate a myriad of processes that occur periodically, from sleeping and waking to how cells use nutrients and energy. One such clock is the one that controls intestinal movements and defecation in the nematode worm Caenorhabditis elegans, which consists of three muscle contractions occurring every 50 seconds. This rhythm is controlled by calcium and proton signalling in the cells of the intestine. The cells of the nematode intestine form a tube, through which gut contents pass. The inside of the tube is acidic, but acidity also plays a role on the outer face of the intestinal tube. In this area, nutrients are distributed and signals are conveyed to other tissues, such as muscles. In fact, acid ­ in the form of protons ­ secreted from the intestinal cells stimulates the muscles that contract in the biological clock that controls the worms' defecation. However, it is poorly understood how the worms control the release of these protons. Kaulich et al. identified two ion channels on the membranes of intestinal cells that become inhibited when the levels of acid surrounding them are high. These channels play distinct roles in controlling the contractions that move the contents of the roundworms' intestines along. The first channel contains a protein called ACD-5, and it is in the membrane of the intestinal cells that faces the inside of the intestinal tube. The second channel is formed by three proteins: FLR-1, ACD-3 and DEL-5. This channel is found on the other side of the intestinal cells, the region where nutrients are distributed and signals are conveyed to the rest of the body. To determine the role of each channel, Kaulich et al. genetically engineered the worms so they would not make the proteins that make up the channels, and imaged the live nematodes to see the effects of removing each channel. The inside of the intestines of worms lacking the ACD-5 containing channel was less acidic than that of normal worms, and the timing of the contractions that control defecation was also slightly altered. Removing the second channel (the one formed by three different proteins), however, had more dramatic effects: the worms were thin, developed more slowly, had less fat tissue and defecated very irregularly. Kaulich et al. imaged live worms to show that the second channel plays a major role in regulating oscillations in acidity both inside and outside cells, as well as controlling calcium levels. This demonstrates that this channel is responsible for the rhythmicity in the contractions that control defecation in the nematodes. Their findings provide important insights towards better understanding proton signalling and the role of acid-sensing ion channels in cellular contexts and biological clocks.

The Caenorhabditis elegans tmc-1 is involved in egg-laying inhibition in response to harsh touch.

The conserved family of Transmembrane channel-like (TMC) proteins has attracted significant interest since two members appear to be key components of the mammalian hair cell mechanotransducer involved in hearing. C. elegans expresses two TMC proteins, TMC-1 and TMC-2. TMC-1 is widely expressed in in both muscles and the nervous system. This wide expression pattern suggests that TMC-1 might serve different functions in the various neurons. TMC-1 has previously been shown to function in neurons, playing a role in chemosensation in the ASH neurons and mechanosensation in OLQ neurons, further supporting this hypothesis. tmc-1 is expressed in the high-threshold mechanosensory neuron, ALA. We show that tmc-1 mutants show defects in the ALA-dependent inhibition of egg-laying in response to a harsh mechanical stimulus.

Chemosensory Neurons Modulate the Response to Oomycete Recognition in Caenorhabditis elegans.

Understanding how animals detect and respond to pathogen threats is central to dissecting mechanisms of host immunity. The oomycetes represent a diverse eukaryotic group infecting various hosts from nematodes to humans. We have previously shown that Caenorhabditis elegans mounts a defense response consisting of the induction of chitinase-like (chil) genes in the epidermis to combat infection by its natural oomycete pathogen Myzocytiopsis humicola. We provide here evidence that C. elegans can sense the oomycete by detecting an innocuous extract derived from animals infected with M. humicola. The oomycete recognition response (ORR) leads to changes in the cuticle and reduction in pathogen attachment, thereby increasing animal survival. We also show that TAX-2/TAX-4 function in chemosensory neurons is required for the induction of chil-27 in the epidermis in response to extract exposure. Our findings highlight that neuron-to-epidermis communication may shape responses to oomycete recognition in animal hosts.

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.

It is all about the support - The role of the extracellular matrix in regenerating axon guidance.

Although it is known for long time that the peripheral nervous system has the capacity for self-regeneration, the molecular mechanisms by which Schwann cells and extracellular matrix (ECM) guide the injured axons to regrow along their original path, remains a poorly understood process. Due to the importance of ECM molecules during development, constitutive mutant organisms display increased lethality, therefore, conditional or inducible strategies have been used to increase the survival of the organisms and allow the study of the role of ECM proteins. In a recent report published in Neuron, Isaacman-Beck and colleagues (2015) used these pioneering genetic studies on zebrafish combined with in vivo fluorescent imaging, to investigate the micro-environmental conditions required for targeted regeneration of the dorsal motor nerve of zebrafish larvae after laser-transection. A candidate gene approach targeting lh3 basal laminar collagen substrates revealed that the lh3 substrate col4α5 regulates dorsal nerve regeneration by destabilizing misdirected axons. Col4α5 was upregulated in a small population of lh3 expressing Schwann cells located ventrally and ventro-laterally to the injury site and found to co-localize with the molecule slit guidance ligand 1 (slit1a). Capitalizing on the crucial observations of mistargeted regeneration of dorsal nerves in mutant larvae, they put forward a model in which Schwann cells shape an environment that allows and directs axonal regeneration to their original synaptic target. In the light of Isaacman-Beck and colleagues (2015) findings, we will review how their study contributes to the research field, and comment on its potential implications for promoting nerve regeneration after injury.