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During aging, preservation of locomotion is generally considered an indicator of sustained good health, in elderlies and in animal models. In Caenorhabditis elegans, mutants of the insulin‐IGF‐1 receptor DAF2/IIRc represent a paradigm of healthy aging, as their increased lifespan is accompanied by a delay in age‐related loss of motility. Here, we investigated the DAF‐2/IIRc‐dependent relationship between longevity and motility using an auxin‐inducible degron to trigger tissue‐specific degradation of endogenous DAF‐2/IIRc. As previously reported, inactivation of DAF‐2/IIRc in neurons or intestine was sufficient to extend the lifespan of worms, whereas depletion in epidermis, germline, or muscle was not. However, neither intestinal nor neuronal depletion of DAF‐2/IIRc prevented the age‐related loss of motility. In 1‐day‐old adults, DAF‐2/IIRc depletion in neurons reduced motility in a DAF‐16/FOXO dependent manner, while muscle depletion had no effect. By contrast, DAF‐2 depletion in the muscle of middle‐age animals improved their motility independently of DAF‐16/FOXO but required UNC‐120/SRF. Yet, neuronal or muscle DAF‐2/IIRc depletion both preserved the mitochondria network in aging muscle. Overall, these results show that the motility pattern of daf‐2 mutants is determined by the sequential and opposing impact of neurons and muscle tissues and can be dissociated from the regulation of the lifespan. This work also provides the characterization of a versatile tool to analyze the tissue‐specific contribution of insulin‐like signaling in integrated phenotypes at the whole organism level. In C. elegans, age‐associated regulation of motility by the DAF‐2/insulin‐IGF‐1 receptor is determined by the sequential and opposing impact of neurons and muscle and can be dissociated from the lifespan phenotype. Intestinal and neuronal DAF‐2 activities modulate lifespan, whereas muscle DAF‐2 does not. Neuronal DAF‐2 promotes motility in early adulthood through inhibition of DAF‐16/FOXO, whereas muscle DAF‐2 decreases motility in middle age through inactivation of UNC‐120/SRF.

The clustering of neurotransmitter receptors at appropriate postsynaptic sites is essential for controlling synaptic transmission. While most known mechanisms involve receptor binding with cytoplasmic scaffolds, recent evidence highlights the importance of extracellular interactions that directly target receptors. Using Caenorhabditis elegans , we identified a trans-synaptic complex that involves RIG-5 and ZIG-8, two adhesion molecules of the immunoglobulin (Ig) superfamily and orthologous to Drosophila DIPs and Dprs, and mammalian IgLONs. Our results show that RIG-5 and ZIG-8 are anchored in the pre- and postsynaptic membranes, respectively, and interact in vivo via their first Ig domains. Furthermore, ZIG-8 directly binds a α7-like acetylcholine receptor (AChR), known as ACR-16, via a cis- interaction between its Ig2 domain and the base of the extracellular AChR domain. This study provides direct evidence that trans-synaptic IgLON interactions can organize neurochemical synapses and suggests that the IgLONs may directly interact with ionotropic receptors in the mammalian nervous system. Synaptic extracellular proteins increasingly emerge as key organizers of neurotransmitter receptors. Here, authors show that an IgLON family cell adhesion molecule directly traps a nicotinic receptor through extracellular interactions.

Increasing evidence indicates that guidance molecules used during development for cellular and axonal navigation also play roles in synapse maturation and homeostasis. In C. elegans the netrin receptor UNC-40/DCC controls the growth of dendritic-like muscle cell extensions towards motoneurons and is required to recruit type A GABA receptors (GABA A Rs) at inhibitory neuromuscular junctions. Here we show that activation of UNC-40 assembles an intracellular synaptic scaffold by physically interacting with FRM-3, a FERM protein orthologous to FARP1/2. FRM-3 then recruits LIN-2, the ortholog of CASK, that binds the synaptic adhesion molecule NLG-1/Neuroligin and physically connects GABA A Rs to prepositioned NLG-1 clusters. These processes are orchestrated by the synaptic organizer CePunctin/MADD-4, which controls the localization of GABA A Rs by positioning NLG-1/neuroligin at synapses and regulates the synaptic content of GABA A Rs through the UNC-40-dependent intracellular scaffold. Since DCC is detected at GABA synapses in mammals, DCC might also tune inhibitory neurotransmission in the mammalian brain. The netrin receptor UNC-40/DCC is required to recruit GABA A R at neuromuscular junctions in C. elegans . Here, the authors show that UNC-40/DCC assembles an intracellular synaptic scaffold, regulating the content of GABA A R and inhibitory neurotransmission.

Summary Aging is commonly defined as the loss of global homeostasis, which results from progressive alteration of all organs function. This model is currently challenged by recent data showing that interventions that extend lifespan do not always increase the overall fitness of the organism. These data suggest the existence of tissue‐specific factors that regulate the pace of aging in a cell‐autonomous manner. Here, we investigated aging of Caenorhabditis elegans striated muscles at the subcellular and the physiological level. Our data show that muscle aging is characterized by a dramatic decrease in the expression of genes encoding proteins required for muscle contraction, followed by a change in mitochondria morphology, and an increase in autophagosome number. Myofilaments, however, remain unaffected during aging. We demonstrated that the conserved transcription factor UNC‐120/SRF regulates muscle aging biomarkers. Interestingly, the role of UNC‐120/SRF in the control of muscle aging can be dissociated from its broader effect on lifespan. In daf‐2/insulin/IGF1 receptor mutants, which exhibit a delayed appearance of muscle aging biomarkers and are long‐lived, disruption of unc‐120 accelerates muscle aging but does not suppress the lifespan phenotype of daf‐2 mutant. Conversely, unc‐120 overexpression delays muscle aging but does not increase lifespan. Overall, we demonstrate that UNC‐120/SRF controls the pace of muscle aging in a cell‐autonomous manner downstream of the insulin/IGF1 receptor.

Inhibitory synapses represent a minority of the total chemical synapses in the mammalian brain, yet proper tuning of inhibition is fundamental to shape neuronal network properties. The neurotransmitter γ-aminobutyric acid (GABA) mediates rapid synaptic inhibition by the activation of the type A GABA receptor (GABA R), a pentameric chloride channel that governs major inhibitory neuronal transduction in the nervous system. Impaired GABA transmission leads to a variety of neuropsychiatric diseases, including schizophrenia, autism, epilepsy or anxiety. From an evolutionary perspective, GABA R shows remarkable conservations, and are found in all eukaryotic clades and even in bacteria and archaea. Specifically, GABA Rs are found in the nematode . Because of the anatomical simplicity of the nervous system and its amenability to genetic manipulations, provide a powerful system to investigate the molecular and cellular biology of GABA synapses. In this mini review article, we will introduce the structure of the GABAergic system and describe recent advances that have identified novel proteins controlling the localization of GABA Rs at synapses. In particular, Ce-Punctin/MADD-4 is an evolutionarily-conserved extracellular matrix protein that behaves as an anterograde synaptic organizer to instruct the excitatory or inhibitory identity of postsynaptic domains.

Single-molecule (SM) fluorescence microscopy allows the imaging of biomolecules in cultured cells with a precision of a few nanometres but has yet to be implemented in living adult animals. Here we used split-GFP (green fluorescent protein) fusions and complementation-activated light microscopy (CALM) for subresolution imaging of individual membrane proteins in live Caenorhabditis elegans (C. elegans) . In vivo tissue-specific SM tracking of transmembrane CD4 and voltage-dependent Ca 2+ channels (VDCC) was achieved with a precision of 30 nm within neuromuscular synapses and at the surface of muscle cells in normal and dystrophin-mutant worms. Through diffusion analyses, we reveal that dystrophin is involved in modulating the confinement of VDCC within sarcolemmal membrane nanodomains in response to varying tonus of C. elegans body-wall muscles. CALM expands the applications of SM imaging techniques beyond the petri dish and opens the possibility to explore the molecular basis of homeostatic and pathological cellular processes with subresolution precision, directly in live animals. Single molecule fluorescence microscopy is a powerful technique to study protein dynamics in cells, but it has not been applied to adult animals. The authors use complementation-activated light microscopy in C. elegans to discover that dystrophin regulates the diffusion properties of voltage-dependent calcium ion channels at the surface of body-wall muscle cells.

The number of nicotinic acetylcholine receptors (AChRs) present in the plasma membrane of muscle and neuronal cells is limited by the assembly of individual subunits into mature pentameric receptors. This process is usually inefficient, and a large number of the synthesized subunits are degraded by endoplasmic reticulum (ER)-associated degradation. To identify cellular factors required for the synthesis of AChRs, we performed a genetic screen in the nematode Caenorhabditis elegans for mutants with decreased sensitivity to the cholinergic agonist levamisole. We isolated a partial loss-of-function allele of ER membrane protein complex-6 (emc-6) , a previously uncharacterized gene in C. elegans . emc-6 encodes an evolutionarily conserved 111-aa protein with two predicted transmembrane domains. EMC-6 is ubiquitously expressed and localizes to the ER. Partial inhibition of EMC-6 caused decreased expression of heteromeric levamisole-sensitive AChRs by destabilizing unassembled subunits in the ER. Inhibition of emc-6 also reduced the expression of homomeric nicotine-sensitive AChRs and GABA A receptors in C. elegans muscle cells. emc-6 is orthologous to the yeast and human EMC6 genes that code for a component of the recently identified ER membrane complex (EMC). Our data suggest this complex is required for protein folding and is connected to ER-associated degradation. We demonstrated that inactivation of additional EMC members in C. elegans also impaired AChR synthesis and induced the unfolded protein response. These results suggest that the EMC is a component of the ER folding machinery. AChRs might provide a valuable proxy to decipher the function of the EMC further.

The assembly of neurotransmitter receptors in the endoplasmic reticulum limits the number of receptors delivered to the plasma membrane, ultimately controlling neurotransmitter sensitivity and synaptic transfer function. In a forward genetic screen conducted in the nematode C. elegans, we identified crld-1 as a gene required for the synaptic expression of ionotropic acetylcholine receptors (AChR). We demonstrated that the CRLD-1A isoform is a membrane-associated ER-resident protein disulfide isomerase (PDI). It physically interacts with AChRs and promotes the assembly of AChR subunits in the ER. Mutations of Creld1, the human ortholog of crld-1a, are responsible for developmental cardiac defects. We showed that Creld1 knockdown in mouse muscle cells decreased surface expression of AChRs and that expression of mouse Creld1 in C. elegans rescued crld-1a mutant phenotypes. Altogether these results identify a novel and evolutionarily-conserved maturational enhancer of AChR biogenesis, which controls the abundance of functional receptors at the cell surface.

At Caenorhabditis elegans neuromuscular junctions (NMJs), synaptic clustering of the levamisole‐sensitive acetylcholine receptors (L‐AChRs) relies on an extracellular scaffold assembled in the synaptic cleft. It involves the secreted protein LEV‐9 and the ectodomain of the transmembrane protein LEV‐10, which are both expressed by muscle cells. L‐AChRs, LEV‐9 and LEV‐10 are part of a physical complex, which localizes at NMJs, yet none of its components localizes independently at synapses. In a screen for mutants partially resistant to the cholinergic agonist levamisole, we identified oig ‐ 4 , which encodes a small protein containing a single immunoglobulin domain. The OIG‐4 protein is secreted by muscle cells and physically interacts with the L‐AChR/LEV‐9/LEV‐10 complex. Removal of OIG‐4 destabilizes the complex and causes a loss of L‐AChR clusters at the synapse. Interestingly, OIG‐4 partially localizes at NMJs independently of LEV‐9 and LEV‐10, thus providing a potential link between the L‐AChR‐associated scaffold and local synaptic cues. These results add a novel paradigm for the immunoglobulin super‐family as OIG‐4 is a secreted protein required for clustering ionotropic receptors independently of synapse formation. Clustering of acetylcholine receptors (L‐AChRs) in Caenorhabditis elegans neuromuscular junctions depends upon on an extracellular scaffold assembled in the synaptic cleft. Here, the findings identify a new component of this scaffold and show that the secreted protein OIG‐4 regulates L‐AChR clustering at the synapse.

Background/Objectives: Pathogenic variants in GNAO1, encoding the inhibitory G protein subunit Gαo, cause severe neurodevelopmental disorders that remain largely refractory to pharmacological treatments. Gαo transduces inhibitory signals downstream of multiple G protein-coupled receptors (GPCRs) involved in motor control. Here, we used gene-edited Caenorhabditis elegans models carrying goa-1 variants, the ortholog of GNAO1, to investigate GPCR contributions to Gαo-dependent locomotor phenotypes. Methods: We combined pharmacological screening of dopamine- and cannabinoid-targeting ligands in goa-1 mutants with structural analysis of ligand-binding pocket conservation and genetic perturbation of receptor function using RNAi and knockout approaches. Results: Pharmacological modulation of GPCR signaling produced non-linear and context-dependent effects. Compounds predicted to further increase excitability may instead promote phenotypic improvement, consistent with compensatory network rebalancing. Structural analyses revealed substantial divergence in ligand-binding pocket conservation for several GPCR-ligand pairs, suggesting that altered binding affinity and selectivity may also contribute to the observed phenotypic outcome. Pharmacological experiments performed in GPCR-depleted mutants allowed for the correlation of structural findings with functional effects for selected receptor-ligand pairs. Finally, genetic reduction in GPCRs coupled to stimulatory G proteins ameliorated hyperactive locomotion in goa-1 mutants, whereas reduction in GPCRs coupled to inhibitory G proteins is largely insufficient to induce or exacerbate locomotor defects. Conclusions: Our findings identify excessive excitatory GPCR input as a key modulator of motor dysfunction in the context of impaired Gαo signaling. They also show that structural conservation is a necessary but not sufficient condition to predict functional responses. Overall, this study establishes C. elegans as a suitable platform to dissect GPCR-mediated signaling and highlights the value of integrating pharmacological and genetic approaches to guide target selection in GNAO1-related disorders.