Génétique et neurobiologie de C. elegans

Pharmacological adaptation is a common phenomenon observed during prolonged drug exposure and often leads to drug resistance. Understanding the cellular events involved in adaptation could provide new strategies to circumvent this resistance issue. We used the nematode Caenorhabditis elegans to analyze the adaptation to levamisole, an ionotropic acetylcholine receptor agonist, used for decades to treat nematode parasitic infections. Genetic screens in C. elegans identified 'adapting mutants' that initially paralyze upon exposure to levamisole as the wild type (WT), but recover locomotion after a few hours whereas WT remain paralyzed. Here, we show that levamisole induces a sustained increase in cytosolic calcium concentration in the muscle cells of adapting mutants, lasting several hours and preceding a decrease in levamisole-sensitive acetylcholine receptors (L-AChR) at the muscle plasma membrane. This decrease correlated with a drop in calcium concentration, a relaxation of the animal's body and a resumption of locomotion. The decrease in calcium and L-AChR content depends on calcineurin activation in muscle cells. We also showed that levamisole adaptation triggers homeostatic mechanisms in muscle cells including mitochondria remodeling, lysosomal tubulation and an increase in autophagic activity. Levamisole adaptation thus provides a new experimental paradigm for studying how cells cope with calcium stress.

The nematode Caenorhabditis elegans is a research model organism particularly suited to the mechanistic understanding of synapse genesis in the nervous system. Armed with powerful genetics, knowledge of complete connectomics, and modern genomics, studies using C. elegans have unveiled multiple key regulators in the formation of a functional synapse. Importantly, many signaling networks display remarkable conservation throughout animals, underscoring the contributions of C. elegans research to advance the understanding of our brain. In this chapter, we will review up-to-date information of the contribution of C. elegans to the understanding of chemical synapses, from structure to molecules and to synaptic remodeling.

The molecular code that controls synapse formation and maintenance in vivo has remained quite sparse. Here, we identify that the secreted protein Adamtsl3 functions as critical hippocampal synapse organizer acting through the transmembrane receptor DCC (deleted in colorectal cancer). Traditionally, DCC function has been associated with glutamatergic synaptogenesis and plasticity in response to Netrin-1 signaling. We demonstrate that early post-natal deletion of Adamtsl3 in neurons impairs DCC protein expression, causing reduced density of both glutamatergic and GABAergic synapses. Adult deletion of Adamtsl3 in either GABAergic or glutamatergic neurons does not interfere with DCC-Netrin-1 function at glutamatergic synapses but controls DCC signaling at GABAergic synapses. The Adamtsl3-DCC signaling unit is further essential for activity-dependent adaptations at GABAergic synapses, involving DCC phosphorylation and Src kinase activation. These findings might be particularly relevant for schizophrenia because genetic variants in Adamtsl3 and DCC have been independently linked with schizophrenia in patients.

The appearance of synapses was a crucial step in the creation of the variety of nervous systems that are found in the animal kingdom. With increased complexity of the organisms came a greater number of synaptic proteins. In this review we describe synaptic proteins that contain the structural domains CUB, CCP, or TSP-1. These domains are found in invertebrates and vertebrates, and CUB and CCP domains were initially described in proteins belonging to the complement system of innate immunity. Interestingly, they are found in synapses of the nematode C. elegans, which does not have a complement system, suggesting an ancient function. Comparison of the roles of CUB-, CCP-, and TSP-1 containing synaptic proteins in various species shows that in more complex nervous systems, these structural domains are combined with other domains and that there is partial conservation of their function. These three domains are thus basic building blocks of the synaptic architecture. Further studies of structural domains characteristic of synaptic proteins in invertebrates such as C. elegans and comparison of their role in mammals will help identify other conserved synaptic molecular building blocks. Furthermore, this type of functional comparison across species will also identify structural domains added during evolution in correlation with increased complexity, shedding light on mechanisms underlying cognition and brain diseases.

Biophysical properties of ligand-gated receptors can be profoundly modified by auxiliary subunits or by the lipid microenvironment of the membrane. Hence, it is sometimes challenging to relate the properties of receptors reconstituted in heterologous expression systems to those of their native counterparts. Here we show that the properties of Caenorhabditis elegans levamisole-sensitive acetylcholine receptors (L-AChRs), the ionotropic acetylcholine receptors targeted by the cholinergic anthelmintic levamisole at neuromuscular junctions, can be profoundly modified by their clustering machinery. We uncovered that L-AChRs exhibit a strong outward rectification in vivo, which was not previously described in heterologous systems. This unusual feature for an ionotropic AChR is abolished by disrupting the interaction of the receptors with the extracellular complex required for their synaptic clustering. When recorded at -60 mV, levamisole-induced currents are similar in the wild type and in L-AChR-clustering-defective mutants, while they are halved in these mutants at more depolarized physiological membrane potentials. Consequently, levamisole causes a strong muscle depolarization in the wild type, which leads to complete inactivation of the voltage-gated calcium channels and to an irreversible flaccid paralysis. In mutants defective for L-AChR clustering, the levamisole-induced depolarization is weaker, allowing voltage-gated calcium channels to remain partially active, which eventually leads to adaptation and survival of the worms. This explains why historical screens for C. elegans mutants resistant to levamisole identified the components of the L-AChR clustering machinery, in addition to proteins required for receptor biosynthesis or efficacy. This work further emphasizes the importance of pursuing ligand-gated channel characterization in their native environment.

Heparan sulfate (HS) proteoglycans contribute to the structural organization of various neurochemical synapses. Depending on the system, their role involves either the core protein or the glycosaminoglycan chains. These linear sugar chains are extensively modified by HS modification enzymes, resulting in highly diverse molecules. Specific modifications of glycosaminoglycan chains may thus contribute to a sugar code involved in synapse specificity. Caenorhabditis elegans is particularly useful to address this question because of the low level of genomic redundancy of these enzymes, as opposed to mammals. Here, we systematically mutated the genes encoding HS modification enzymes in C. elegans and analyzed their impact on excitatory and inhibitory neuromuscular junctions (NMJs). Using single chain antibodies that recognize different HS modification patterns, we show in vivo that these two HS epitopes are carried by the SDN-1 core protein, the unique C. elegans syndecan ortholog, at NMJs. Intriguingly, these antibodies differentially bind to excitatory and inhibitory synapses, implying unique HS modification patterns at different NMJs. Moreover, while most enzymes are individually dispensable for proper organization of NMJs, we show that 3-O-sulfation of SDN-1 is required to maintain wild-type levels of the extracellular matrix protein MADD-4/Punctin, a central synaptic organizer that defines the identity of excitatory and inhibitory synaptic domains at the plasma membrane of muscle cells.

The extracellular matrix has emerged as an active component of chemical synapses regulating synaptic formation, maintenance, and homeostasis. The heparan sulfate proteoglycan (HSPG) syndecans are known to regulate cellular and axonal migration in the brain. They are also enriched at synapses, but their synaptic functions remain more elusive. Here, we show that SDN-1, the sole orthologue of syndecan in C. elegans, is absolutely required for the synaptic clustering of homomeric α7-like acetylcholine receptors (AChRs) and regulates the synaptic content of heteromeric AChRs. SDN-1 is concentrated at neuromuscular junctions (NMJs) by the neurally secreted synaptic organizer Ce-Punctin/MADD-4, which also activates the transmembrane netrin receptor DCC. Those cooperatively recruit the FARP and CASK orthologues that localize α7-like-AChRs at cholinergic NMJs through physical interactions. Therefore, SDN-1 stands at the core of the cholinergic synapse organization by bridging the extracellular synaptic determinants to the intracellular synaptic scaffold that controls the postsynaptic receptor content.

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 (GABAARs) 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 GABAARs to prepositioned NLG-1 clusters. These processes are orchestrated by the synaptic organizer CePunctin/MADD-4, which controls the localization of GABAARs by positioning NLG-1/neuroligin at synapses and regulates the synaptic content of GABAARs 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 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.

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.

Diffuse extrasynaptic neurotransmitter receptors constitute an abundant pool of receptors that can be recruited to modulate synaptic strength. Whether the diffuse distribution of receptors in extrasynaptic membranes is a default state or is actively controlled remains essentially unknown. Here we show that RSU-1 (Ras Suppressor-1) is required for the proper distribution of extrasynaptic acetylcholine receptors (AChRs) in Caenorhabditis elegans muscle cells. RSU-1 is an evolutionary conserved cytoplasmic protein that contains multiple leucine-rich repeats (LRRs) and interacts with integrin-dependent adhesion complexes. In rsu-1 mutants, neuromuscular junctions differentiate as in the wild type, but AChRs assemble into ectopic clusters that progressively enlarge during development. As a consequence, the synaptic content of AChRs is reduced. Our study provides the first evidence that an RSU-1-dependent active mechanism maintains extrasynaptic receptors dispersed and indirectly regulates synapse maturation.

Positioning type A GABA receptors (GABA(A)Rs) in front of GABA release sites sets the strength of inhibitory synapses. The evolutionarily conserved Ce-Punctin/MADD-4 is an anterograde synaptic organizer that specifies GABAergic versus cholinergic identity of postsynaptic domains at the C. elegans neuromuscular junctions (NMJs). Here we show that the Ce-Punctin secreted by GABAergic motor neurons controls the clustering of GABA(A)Rs through the synaptic adhesion molecule neuroligin (NLG-1) and the netrin receptor UNC-40/DCC. The short isoform of Ce-Punctin binds and clusters NLG-1 postsynaptically at GABAergic NMJs. NLG-1 disruption causes a strong reduction of GABA(A)R content at GABAergic synapses. Ce-Punctin also binds and localizes UNC-40 receptors in the postsynaptic membrane of NMJs, which promotes the recruitment of GABA(A)Rs by NLG-1. Since the mammalian orthologs of these genes are expressed in the central nervous system and their mutations are implicated in neuropsychiatric diseases, this molecular pathway might have been evolutionarily conserved.

During nervous system development, postmitotic neurons face the challenge of generating and structurally organizing specific synapses with appropriate synaptic partners. An important unexplored question is whether the process of synaptogenesis is coordinated with the adoption of specific signaling properties of a neuron. Such signaling properties are defined by the neurotransmitter system that a neuron uses to communicate with postsynaptic partners, the neurotransmitter receptor type used to receive input from presynaptic neurons, and, potentially, other sensory receptors that activate a neuron. Elucidating the mechanisms that coordinate synaptogenesis, neuronal activation, and neurotransmitter signaling in a postmitotic neuron represents one key approach to understanding how neurons develop as functional units. Using the SAB class of Caenorhabditis elegans motor neurons as a model system, we show here that the phylogenetically conserved COE-type transcription factor UNC-3 is required for synaptogenesis. UNC-3 directly controls the expression of the ADAMTS-like protein MADD-4/Punctin, a presynaptically secreted synapse-organizing molecule that clusters postsynaptic receptors. UNC-3 also controls the assembly of presynaptic specializations and ensures the coordinated expression of enzymes and transporters that define the cholinergic neurotransmitter identity of the SAB neurons. Furthermore, synaptic output properties of the SAB neurons are coordinated with neuronal activation and synaptic input, as evidenced by UNC-3 also regulating the expression of ionotropic neurotransmitter receptors and putative stretch receptors. Our study shows how synaptogenesis and distinct, function-defining signaling features of a postmitotic neuron are hardwired together through coordinated transcriptional control.

Because most neurons receive thousands of synaptic inputs, the neuronal membrane is a mosaic of specialized microdomains where neurotransmitter receptors cluster in register with the corresponding presynaptic neurotransmitter release sites. In many cases the coordinated differentiation of presynaptic and postsynaptic domains implicates trans-synaptic interactions between membrane-associated proteins such as neurexins and neuroligins. The Caenorhabditis elegans neuromuscular junction (NMJ) provides a genetically tractable system in which to analyse the segregation of neurotransmitter receptors, because muscle cells receive excitatory innervation from cholinergic neurons and inhibitory innervation from GABAergic neurons. Here we show that Ce-Punctin/madd-4 (ref. 5), the C. elegans orthologue of mammalian punctin-1 and punctin-2, encodes neurally secreted isoforms that specify the excitatory or inhibitory identity of postsynaptic NMJ domains. These proteins belong to the ADAMTS (a disintegrin and metalloprotease with thrombospondin repeats)-like family, a class of extracellular matrix proteins related to the ADAM proteases but devoid of proteolytic activity. Ce-Punctin deletion causes the redistribution of synaptic acetylcholine and GABAA (γ-aminobutyric acid type A) receptors into extrasynaptic clusters, whereas neuronal presynaptic boutons remain unaltered. Alternative promoters generate different Ce-Punctin isoforms with distinct functions. A short isoform is expressed by cholinergic and GABAergic motoneurons and localizes to excitatory and inhibitory NMJs, whereas long isoforms are expressed exclusively by cholinergic motoneurons and are confined to cholinergic NMJs. The differential expression of these isoforms controls the congruence between presynaptic and postsynaptic domains: specific disruption of the short isoform relocalizes GABAA receptors from GABAergic to cholinergic synapses, whereas expression of a long isoform in GABAergic neurons recruits acetylcholine receptors to GABAergic NMJs. These results identify Ce-Punctin as a previously unknown synaptic organizer and show that presynaptic and postsynaptic domain identities can be genetically uncoupled in vivo. Because human punctin-2 was identified as a candidate gene for schizophrenia, ADAMTS-like proteins may also control synapse organization in the mammalian central nervous system.

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 GABAA 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.

Auxiliary subunits regulate the trafficking, localization or gating kinetics of voltage- and ligand-gated ion channels by associating tightly and specifically with pore-forming subunits. However, no auxiliary subunits have been identified for members of the Cys-loop receptor superfamily. Here we identify MOLO-1, a positive regulator of levamisole-sensitive acetylcholine receptors (L-AChRs) at the Caenorhabditis elegans neuromuscular junction. MOLO-1 is a one-pass transmembrane protein that contains a single extracellular globular domain-the TPM domain, found in bacteria, plants and invertebrates, including nonvertebrate chordates. Loss of MOLO-1 impairs locomotion and renders worms resistant to the anthelmintic drug levamisole. In molo-1 mutants, L-AChR-dependent synaptic transmission is reduced by half, while the number and localization of receptors at synapses remain unchanged. In a heterologous expression system, MOLO-1 physically interacts with L-AChRs and directly enhances channel gating without affecting unitary conductance. The identification of MOLO-1 expands the mechanisms for generating functional and pharmacological diversity in the Cys-loop superfamily.

Efficient neurotransmission at chemical synapses relies on spatial congruence between the presynaptic active zone, where synaptic vesicles fuse, and the postsynaptic differentiation, where neurotransmitter receptors concentrate. Diverse molecular systems have evolved to localize receptors at synapses, but in most cases, they rely on scaffolding proteins localized below the plasma membrane. A few systems have been suggested to control the synaptic localization of neurotransmitter receptors through extracellular interactions, such as the pentraxins that bind AMPA receptors and trigger their aggregation. However, it is not yet clear whether these systems have a central role in the organization of postsynaptic domains in vivo or rather provide modulatory functions. Here we describe an extracellular scaffold that is necessary to cluster acetylcholine receptors at neuromuscular junctions in the nematode Caenorhabditis elegans. It involves the ectodomain of the previously identified transmembrane protein LEV-10 (ref. 6) and a novel extracellular protein, LEV-9. LEV-9 is secreted by the muscle cells and localizes at cholinergic neuromuscular junctions. Acetylcholine receptors, LEV-9 and LEV-10 are interdependent for proper synaptic localization and physically interact based on biochemical evidence. Notably, the function of LEV-9 relies on eight complement control protein (CCP) domains. These domains, also called 'sushi domains', are usually found in proteins regulating complement activity in the vertebrate immune system. Because the complement system does not exist in protostomes, our results suggest that some of the numerous uncharacterized CCP proteins expressed in the mammalian brain might be directly involved in the organization of the synapse, independently from immune functions.

Levamisole-sensitive acetylcholine receptors (L-AChRs) are ligand-gated ion channels that mediate excitatory neurotransmission at the neuromuscular junctions of nematodes. They constitute a major drug target for anthelminthic treatments because they can be activated by nematode-specific cholinergic agonists such as levamisole. Genetic screens conducted in Caenorhabditis elegans for resistance to levamisole toxicity identified genes that are indispensable for the biosynthesis of L-AChRs. These include 5 genes encoding distinct AChR subunits and 3 genes coding for ancillary proteins involved in assembly and trafficking of the receptors. Despite extensive analysis of L-AChRs in vivo, pharmacological and biophysical characterization of these receptors has been greatly hampered by the absence of a heterologous expression system. Using Xenopus laevis oocytes, we were able to reconstitute functional L-AChRs by coexpressing the 5 distinct receptor subunits and the 3 ancillary proteins. Strikingly, this system recapitulates the genetic requirements for receptor expression in vivo because omission of any of these 8 genes dramatically impairs L-AChR expression. We demonstrate that 3 alpha- and 2 non-alpha-subunits assemble into the same receptor. Pharmacological analysis reveals that the prototypical cholinergic agonist nicotine is unable to activate L-AChRs but rather acts as a potent allosteric inhibitor. These results emphasize the role of ancillary proteins for efficient expression of recombinant neurotransmitter receptors and open the way for in vitro screening of novel anthelminthic agents.

Clustering neurotransmitter receptors at the synapse is crucial for efficient neurotransmission. Here we identify a Caenorhabditis elegans locus, lev-10, required for postsynaptic aggregation of ionotropic acetylcholine receptors (AChRs). lev-10 mutants were identified on the basis of weak resistance to the anthelminthic drug levamisole, a nematode-specific cholinergic agonist that activates AChRs present at neuromuscular junctions (NMJs) resulting in muscle hypercontraction and death at high concentrations. In lev-10 mutants, the density of levamisole-sensitive AChRs at NMJs is markedly reduced, yet the number of functional AChRs present at the muscle cell surface remains unchanged. LEV-10 is a transmembrane protein localized to cholinergic NMJs and required in body-wall muscles for AChR clustering. We also show that the LEV-10 extracellular region, containing five predicted CUB domains and one LDLa domain, is sufficient to rescue AChR aggregation in lev-10 mutants. This suggests a mechanism for AChR clustering that relies on extracellular protein-protein interactions. Such a mechanism is likely to be evolutionarily conserved because CUB/LDL transmembrane proteins similar to LEV-10, but lacking any assigned function, are expressed in the mammalian nervous system and might be used to cluster ionotropic receptors in vertebrates.