A serotonergic circuit regulates aversive associative learning under mitochondrial stress in C. elegans.

SignificancePhysiological stress triggers avoidance behavior, allowing the animals to stay away from potential threats and optimize their chance of survival. Mitochondrial disruption, a common physiological stress in diverse species, induces the nematode Caenorhabditis elegans to avoid non-pathogenic bacteria through a serotonergic neuronal circuit. We find that distinct neurons, communicated through serotonin and a specific serotonin receptor, are required for the formation and retrieval of this learned aversive behavior. This learned avoidance behavior is associated with increased serotonin synthesis, altered neuronal response property, and reprogramming of locomotion patterns. The circuit and neuromodulatory mechanisms described here offer important insights for stress-induced avoidance behavior.

Cell polarity control by Wnt morphogens.

Cell polarity is regulated by both intrinsic properties of the cell and extrinsic factors in the environment. Wnts are secreted glycoproteins in graded distribution, and they function as morphogens to instruct cell fate and as guidance cues to steer axon growth cone, respectively. Recent studies suggest that Wnts also instruct cell polarization in diverse contexts, by engaging cytoskeletal machineries or transcriptional mechanisms. Here we review the literature of cell polarity control by Wnt glycoproteins, with an emphasis on the nematode Caenorhabditis elegans, a multi-cellular organism in which the importance of polarity-inducing factors can be verified in vivo. In both embryonic and postembryonic cell lineages that undergo asymmetric division, Wnts act as directional signals to instruct the asymmetry of mitosis. In C. elegans, Wnts polarize neuroblasts to control their directional migration, and they also specify axon-dendrite polarity by providing spatial instruction for postmitotic neurons. Together this review summarizes recent advances and unsolved issues in cell polarity control by Wnt glycoproteins.

Caenorhabditis elegans Flamingo FMI-1 controls dendrite self-avoidance through F-actin assembly.

Self-avoidance is a conserved mechanism that prevents crossover between sister dendrites from the same neuron, ensuring proper functioning of the neuronal circuits. Several adhesion molecules are known to be important for dendrite self-avoidance, but the underlying molecular mechanisms are incompletely defined. Here, we show that FMI-1/Flamingo, an atypical cadherin, is required autonomously for self-avoidance in the multidendritic PVD neuron of Caenorhabditis elegans The fmi-1 mutant shows increased crossover between sister PVD dendrites. Our genetic analysis suggests that FMI-1 promotes transient F-actin assembly at the tips of contacting sister dendrites to facilitate their efficient retraction during self-avoidance events, probably by interacting with WSP-1/N-WASP. Mutations of vang-1, which encodes the planar cell polarity protein Vangl2 previously shown to inhibit F-actin assembly, suppress self-avoidance defects of the fmi-1 mutant. FMI-1 downregulates VANG-1 levels probably through forming protein complexes. Our study identifies molecular links between Flamingo and the F-actin cytoskeleton that facilitate efficient dendrite self-avoidance.

Wnt signalling and endocytosis: Mechanisms, controversies and implications for stress responses.

Wnt signalling is one of a few conserved pathways that control diverse aspects of development and morphogenesis in all metazoan species. Endocytosis is a key mechanism that regulates the secretion and graded extracellular distribution of Wnt glycoproteins from the source cells, as well as Wnt signal transduction in the receiving cells. However, controversies exist regarding the requirement of clathrin-dependent endocytosis in Wnt signalling. Various lines of evidence from recent studies suggest that Wnt-ß-catenin signalling is also involved in the regulation of cellular stress responses in adulthood, a role that is beyond its canonical functions in animal development. In this review, we summarise recent advances in the molecular and cellular mechanisms by which endocytosis modulates Wnt signalling. We also discuss how Wnt signalling could be repurposed to regulate mitochondrial stress response in the nematode Caenorhabditis elegans.

The polarity protein VANG-1 antagonizes Wnt signaling by facilitating Frizzled endocytosis.

Signaling that instructs the migration of neurons needs to be tightly regulated to ensure precise positioning of neurons and subsequent wiring of the neuronal circuits. Wnt-Frizzled signaling controls neuronal migration in metazoans, in addition to many other aspects of neural development. We show that Caenorhabditis elegans VANG-1, a membrane protein that acts in the planar cell polarity (PCP) pathway, antagonizes Wnt signaling by facilitating endocytosis of the Frizzled receptors. Mutations of vang-1 suppress migration defects of multiple classes of neurons in the Frizzled mutants, and overexpression of vang-1 causes neuronal migration defects similar to those of the Frizzled mutants. Our genetic experiments suggest that VANG-1 facilitates Frizzled endocytosis through ß-arrestin2. Co-immunoprecipitation experiments indicate that Frizzled proteins and VANG-1 form a complex, and this physical interaction requires the Frizzled cysteine-rich domain. Our work reveals a novel mechanism mediated by the PCP protein VANG-1 that downregulates Wnt signaling through Frizzled endocytosis.

A Wnt-planar polarity pathway instructs neurite branching by restricting F-actin assembly through endosomal signaling.

Spatial arrangement of neurite branching is instructed by both attractive and repulsive cues. Here we show that in C. elegans, the Wnt family of secreted glycoproteins specify neurite branching sites in the PLM mechanosensory neurons. Wnts function through MIG-1/Frizzled and the planar cell polarity protein (PCP) VANG-1/Strabismus/Vangl2 to restrict the formation of F-actin patches, which mark branching sites in nascent neurites. We find that VANG-1 promotes Wnt signaling by facilitating Frizzled endocytosis and genetically acts in a common pathway with arr-1/ß-arrestin, whose mutation results in defective PLM branching and F-actin patterns similar to those in the Wnt, mig-1 or vang-1 mutants. On the other hand, the UNC-6/Netrin pathway intersects orthogonally with Wnt-PCP signaling to guide PLM branch growth along the dorsal-ventral axis. Our study provides insights for how attractive and repulsive signals coordinate to sculpt neurite branching patterns, which are critical for circuit connectivity.

Neural activity and CaMKII protect mitochondria from fragmentation in aging Caenorhabditis elegans neurons.

Decline in mitochondrial morphology and function is a hallmark of neuronal aging. Here we report that progressive mitochondrial fragmentation is a common manifestation of aging Caenorhabditis elegans neurons and body wall muscles. We show that sensory-evoked activity was essential for maintaining neuronal mitochondrial morphology, and this activity-dependent mechanism required the Degenerin/ENaC sodium channel MEC-4, the L-type voltage-gated calcium channel EGL-19, and the Ca/calmodulin-dependent kinase II (CaMKII) UNC-43. Importantly, UNC-43 phosphorylated and inhibited the dynamin-related protein (DRP)-1, which was responsible for excessive mitochondrial fragmentation in neurons that lacked sensory-evoked activity. Moreover, enhanced activity in the aged neurons ameliorated mitochondrial fragmentation. These findings provide a detailed description of mitochondrial behavior in aging neurons and identify activity-dependent DRP-1 phosphorylation by CaMKII as a key mechanism in neuronal mitochondrial maintenance.

RHGF-1/PDZ-RhoGEF and retrograde DLK-1 signaling drive neuronal remodeling on microtubule disassembly.

Neurons remodel their connectivity in response to various insults, including microtubule disruption. How neurons sense microtubule disassembly and mount remodeling responses by altering genetic programs in the soma are not well defined. Here we show that in response to microtubule disassembly, the Caenorhabditis elegans PLM neuron remodels by retracting its synaptic branch and overextending the primary neurite. This remodeling required RHGF-1, a PDZ-Rho guanine nucleotide exchange factor (PDZ-RhoGEF) that was associated with and inhibited by microtubules. Independent of the myosin light chain activation, RHGF-1 acted through Rho-dependent kinase LET-502/ROCK and activated a conserved, retrograde DLK-1 MAPK (DLK-1/dual leucine zipper kinase) pathway, which triggered synaptic branch retraction and overgrowth of the PLM neurite in a dose-dependent manner. Our data represent a neuronal remodeling paradigm during development that reshapes the neural circuit by the coordinated removal of the dysfunctional synaptic branch compartment and compensatory extension of the primary neurite.

Genetic analysis of a novel tubulin mutation that redirects synaptic vesicle targeting and causes neurite degeneration in C. elegans.

Neuronal cargos are differentially targeted to either axons or dendrites, and this polarized cargo targeting critically depends on the interaction between microtubules and molecular motors. From a forward mutagenesis screen, we identified a gain-of-function mutation in the C. elegans α-tubulin gene mec-12 that triggered synaptic vesicle mistargeting, neurite swelling and neurodegeneration in the touch receptor neurons. This missense mutation replaced an absolutely conserved glycine in the H12 helix with glutamic acid, resulting in increased negative charges at the C-terminus of α-tubulin. Synaptic vesicle mistargeting in the mutant neurons was suppressed by reducing dynein function, suggesting that aberrantly high dynein activity mistargeted synaptic vesicles. We demonstrated that dynein showed preference towards binding mutant microtubules over wild-type in microtubule sedimentation assay. By contrast, neurite swelling and neurodegeneration were independent of dynein and could be ameliorated by genetic paralysis of the animal. This suggests that mutant microtubules render the neurons susceptible to recurrent mechanical stress induced by muscle activity, which is consistent with the observation that microtubule network was disorganized under electron microscopy. Our work provides insights into how microtubule-dynein interaction instructs synaptic vesicle targeting and the importance of microtubule in the maintenance of neuronal structures against constant mechanical stress.

Genetic analysis of age-dependent defects of the Caenorhabditis elegans touch receptor neurons.

Although many genes have been implicated in the pathogenesis of common neurodegenerative diseases, the genetic and cellular mechanisms that maintain neuronal integrity during normal aging remain elusive. Here we show that Caenorhabditis elegans touch receptor and cholinergic neurons display age-dependent morphological defects, including cytoskeletal disorganization, axon beading, and defasciculation. Progression of neuronal aging is regulated by DAF-2 and DAF-16 signaling, which also modulate adult life span. Mutations that disrupt touch-evoked sensory activity or reduce membrane excitability trigger accelerated neuronal aging, indicating that electrical activity is critical for adult neuronal integrity. Disrupting touch neuron attachment to the epithelial cells induces distinct neurodegenerative phenotypes. These results provide a detailed description of the age-dependent morphological defects that occur in identified neurons of C. elegans, demonstrate that the age of onset of these defects is regulated by specific genes, and offer experimental evidence for the importance of normal levels of neural activity in delaying neuronal aging.

Peripheral peroxisomal ß-oxidation engages neuronal serotonin signaling to drive stress-induced aversive memory in C. elegans.

Physiological dysfunction confers negative valence to coincidental sensory cues to induce the formation of aversive associative memory. How peripheral tissue stress engages neuromodulatory mechanisms to form aversive memory is poorly understood. Here, we show that in the nematode C. elegans, mitochondrial disruption induces aversive memory through peroxisomal ß-oxidation genes in non-neural tissues, including pmp-4/very-long-chain fatty acid transporter, dhs-28/3-hydroxylacyl-CoA dehydrogenase, and daf-22/3-ketoacyl-CoA thiolase. Upregulation of peroxisomal ß-oxidation genes under mitochondrial stress requires the nuclear hormone receptor NHR-49. Importantly, the memory-promoting function of peroxisomal ß-oxidation is independent of its canonical role in pheromone production. Peripheral signals derived from the peroxisomes target NSM, a critical neuron for memory formation under stress, to upregulate serotonin synthesis and remodel evoked responses to sensory cues. Our genetic, transcriptomic, and metabolomic approaches establish peroxisomal lipid signaling as a crucial mechanism that connects peripheral mitochondrial stress to central serotonin neuromodulation in aversive memory formation.

C. elegans AP-2 and retromer control Wnt signaling by regulating mig-14/Wntless.

While endocytosis can regulate morphogen distribution, its precise role in shaping these gradients is unclear. Even more enigmatic is the role of retromer, a complex that shuttles proteins between endosomes and the Golgi apparatus, in Wnt gradient formation. Here we report that DPY-23, the C. elegans mu subunit of the clathrin adaptor AP-2 that mediates the endocytosis of membrane proteins, regulates Wnt function. dpy-23 mutants display Wnt phenotypes, including defects in neuronal migration, neuronal polarity, and asymmetric cell division. DPY-23 acts in Wnt-expressing cells to promote these processes. MIG-14, the C. elegans homolog of the Wnt-secretion factor Wntless, also acts in these cells to control Wnt function. In dpy-23 mutants, MIG-14 accumulates at or near the plasma membrane. By contrast, MIG-14 accumulates in intracellular compartments in retromer mutants. Based on our observations, we propose that intracellular trafficking of MIG-14 by AP-2 and retromer plays an important role in Wnt secretion.

An olfactory-interneuron circuit that drives stress-induced avoidance behavior in C. elegans.

Physiological stress represents a drastic change of internal state and can drive avoidance behavior, but the neural circuits are incompletely defined. Here, we characterize a sensory-interneuron circuit for mitochondrial stress-induced avoidance behavior in C. elegans. The olfactory sensory neurons and the AIY interneuron are essential, with the olfactory neurons acting upstream of AIY. Unlike pathogen avoidance, stress-induced avoidance does not require AIB, AIZ or RIA interneurons. Ablation or inhibition of the head motor neurons SMDD/V alters the worm's locomotion and reduces avoidance. These findings substantiate our understanding of the circuit mechanisms that underlie learned avoidance behavior triggered by mitochondrial stress.

May the force be with you: Mechanosensitivity shapes dendrite development.

Mechanical stimuli have profound effects on the structure and function of various cells and tissues. In this issue of Developmental Cell, Tao et al. report that mechanosensory ion channels mediate the effects of cell membrane guidance cues on the morphogenesis of neuronal dendrites.

A role for dopamine in C. elegans avoidance behavior induced by mitochondrial stress.

Physiological stress triggers aversive learning that profoundly alters animal behavior. Systemic mitochondrial disruption induces avoidance of C. elegans to non-pathogenic food bacteria. Mutations in cat-2 and dat-1, which control dopamine synthesis and reuptake, respectively, impair this learned bacterial avoidance, suggesting that dopaminergic modulation is essential. Cell-specific rescue experiments indicate that dopamine likely acts from the CEP and ADE neurons to regulate learned bacterial avoidance. We find that mutations in multiple dopamine receptor genes, including dop-1, dop-2 and dop-3, reduced learned bacterial avoidance. Our work reveals a role for dopamine signaling in C. elegans learned avoidance behavior induced by mitochondrial stress.

Neurophysiological basis of stress-induced aversive memory in the nematode Caenorhabditis elegans.

Physiological stress induces aversive memory formation and profoundly impacts animal behavior. In C. elegans, concurrent mitochondrial disruption induces aversion to the bacteria that the animal inherently prefers, offering an experimental paradigm for studying the neural basis of aversive memory. We find that, under mitochondrial stress, octopamine secreted from the RIC modulatory neuron targets the AIY interneuron through the SER-6 receptor to trigger learned bacterial aversion. RIC responds to systemic mitochondrial stress by increasing octopamine synthesis and acts in the formation of aversive memory. AIY integrates sensory information, acts downstream of RIC, and is important for the retrieval of aversive memory. Systemic mitochondrial dysfunction induces RIC responses to bacterial cues that parallel stress induction, suggesting that physiological stress activates latent communication between RIC and the sensory neurons. These findings provide insights into the circuit and neuromodulatory mechanisms underlying stress-induced aversive memory.

Multiple Wnts and frizzled receptors regulate anteriorly directed cell and growth cone migrations in Caenorhabditis elegans.

A set of conserved molecules guides axons along the metazoan dorsal-ventral axis. Recently, Wnt glycoproteins have been shown to guide axons along the anterior-posterior (A/P) axis of the mammalian spinal cord. Here, we show that, in the nematode Caenorhabditis elegans, multiple Wnts and Frizzled receptors regulate the anterior migrations of neurons and growth cones. Three Wnts are expressed in the tail, and at least one of these, EGL-20, functions as a repellent. We show that the MIG-1 Frizzled receptor acts in the neurons and growth cones to promote their migrations and provide genetic evidence that the Frizzleds MIG-1 and MOM-5 mediate the repulsive effects of EGL-20. While these receptors mediate the effects of EGL-20, we find that the Frizzled receptor LIN-17 can antagonize MIG-1 signaling. Our results indicate that Wnts play a key role in A/P guidance in C. elegans and employ distinct mechanisms to regulate different migrations.

Live-cell imaging of PVD dendritic growth cone in post-embryonic C. elegans.

Live-cell imaging analysis provides tremendous information for the study of cellular events such as growth cone migration in neuronal development. Here, we describe a protocol for live-cell imaging of migrating PVD dendritic growth cones in the nematode C. elegans by spinning-disk confocal microscopy. Fluorescently labeled growth cones and cytoskeletal proteins could be continuously observed for 4-6 h in mid-stage larvae. This protocol is suitable for revealing the dynamic molecular and cellular events in dendrite and axon development of C. elegans. For complete details on the use and execution of this protocol, please refer to Chen et al. (2019).

Neuronal mitochondrial dynamics coordinate systemic mitochondrial morphology and stress response to confer pathogen resistance in C. elegans.

Mitochondrial functions across different tissues are regulated in a coordinated fashion to optimize the fitness of an organism. Mitochondrial unfolded protein response (UPRmt) can be nonautonomously elicited by mitochondrial perturbation in neurons, but neuronal signals that propagate such response and its physiological significance remain incompletely understood. Here, we show that in C. elegans, loss of neuronal fzo-1/mitofusin induces nonautonomous UPRmt through multiple neurotransmitters and neurohormones, including acetylcholine, serotonin, glutamate, tyramine, and insulin-like peptides. Neuronal fzo-1 depletion also triggers nonautonomous mitochondrial fragmentation, which requires autophagy and mitophagy genes. Systemic activation of UPRmt and mitochondrial fragmentation in C. elegans via perturbing neuronal mitochondrial dynamics improves resistance to pathogenic Pseudomonas infection, which is supported by transcriptomic signatures of immunity and stress-response genes. We propose that C. elegans surveils neuronal mitochondrial dynamics to coordinate systemic UPRmt and mitochondrial connectivity for pathogen defense and optimized survival under bacterial infection.

Age-dependent changes in response property and morphology of a thermosensory neuron and thermotaxis behavior in Caenorhabditis elegans.

Age-dependent cognitive and behavioral deterioration may arise from defects in different components of the nervous system, including those of neurons, synapses, glial cells, or a combination of them. We find that AFD, the primary thermosensory neuron of Caenorhabditis elegans, in aged animals is characterized by loss of sensory ending integrity, including reduced actin-based microvilli abundance and aggregation of thermosensory guanylyl cyclases. At the functional level, AFD neurons in aged animals are hypersensitive to high temperatures and show sustained sensory-evoked calcium dynamics, resulting in a prolonged operating range. At the behavioral level, senescent animals display cryophilic behaviors that remain plastic to acute temperature changes. Excessive cyclase activity of the AFD-specific guanylyl cyclase, GCY-8, is associated with developmental defects in AFD sensory ending and cryophilic behavior. Surprisingly, loss of the GCY-8 cyclase domain reduces these age-dependent morphological and behavioral changes, while a prolonged AFD operating range still exists in gcy-8 animals. The lack of apparent correlation between age-dependent changes in the morphology or stimuli-evoked response properties of primary sensory neurons and those in related behaviors highlights the importance of quantitative analyses of aging features when interpreting age-related changes at structural and functional levels. Our work identifies aging hallmarks in AFD receptive ending, temperature-evoked AFD responses, and experience-based thermotaxis behavior, which serve as a foundation to further elucidate the neural basis of cognitive aging.