Intermediate filament accumulation can stabilize microtubules in Caenorhabditis elegans motor neurons.

Neural circuits utilize a coordinated cellular machinery to form and eliminate synaptic connections, with the neuronal cytoskeleton playing a prominent role. During larval development of Caenorhabditis elegans, synapses of motor neurons are stereotypically rewired through a process facilitated by dynamic microtubules (MTs). Through a genetic suppressor screen on mutant animals that fail to rewire synapses, and in combination with live imaging and ultrastructural studies, we find that intermediate filaments (IFs) stabilize MTs to prevent synapse rewiring. Genetic ablation of IFs or pharmacological disruption of IF networks restores MT growth and rescues synapse rewiring defects in the mutant animals, indicating that IF accumulation directly alters MT stability. Our work sheds light on the impact of IFs on MT dynamics and axonal transport, which is relevant to the mechanistic understanding of several human motor neuron diseases characterized by IF accumulation in axonal swellings.

C. elegans MAGU-2/Mpp5 homolog regulates epidermal phagocytosis and synapse density.

Synapses are dynamic connections that underlie essential functions of the nervous system. The addition, removal, and maintenance of synapses govern the flow of information in neural circuits throughout the lifetime of an animal. While extensive studies have elucidated many intrinsic mechanisms that neurons employ to modulate their connections, increasing evidence supports the roles of non-neuronal cells, such as glia, in synapse maintenance and circuit function. We previously showed that C. elegans epidermis regulates synapses through ZIG-10, a cell-adhesion protein of the immunoglobulin domain superfamily. Here we identified a member of the Pals1/MPP5 family, MAGU-2, that functions in the epidermis to modulate phagocytosis and the number of synapses by regulating ZIG-10 localization. Furthermore, we used light and electron microscopy to show that this epidermal mechanism removes neuronal membranes from the neuromuscular junction, dependent on the conserved phagocytic receptor CED-1. Together, our study shows that C. elegans epidermis constrains synaptic connectivity, in a manner similar to astrocytes and microglia in mammals, allowing optimized output of neural circuits.

Caenorhabditis elegans flamingo cadherin fmi-1 regulates GABAergic neuronal development.

In a genetic screen for regulators of synaptic morphology, we identified the single Caenorhabditis elegans flamingo-like cadherin fmi-1. The fmi-1 mutants exhibit defective axon pathfinding, reduced synapse number, aberrant synapse size and morphology, as well as an abnormal accumulation of synaptic vesicles at nonsynaptic regions. Although FMI-1 is primarily expressed in the nervous system, it is not expressed in the ventral D-type (VD) GABAergic motorneurons, which are defective in fmi-1 mutants. The axon and synaptic defects of VD neurons could be rescued when fmi-1 was expressed exclusively in non-VD neighboring neurons, suggesting a cell nonautonomous action of FMI-1. FMI-1 protein that lacked its intracellular domain still retained its ability to rescue the vesicle accumulation defects of GABAergic motorneurons, indicating that the extracellular domain was sufficient for this function of FMI-1 in GABAergic neuromuscular junction development. Mutations in cdh-4, a Fat-like cadherin, cause similar defects in GABAergic motorneurons. The cdh-4 is expressed by the VD neurons and seems to function in the same genetic pathway as fmi-1 to regulate GABAergic neuron development. Thus, fmi-1 and cdh-4 cadherins might act together to regulate synapse development and axon pathfinding.

Nuclear pre-mRNA 3'-end processing regulates synapse and axon development in C. elegans.

Nuclear pre-mRNA 3'-end processing is vital for the production of mature mRNA and the generation of the 3' untranslated region (UTR). However, the roles and regulation of this event in cellular development remain poorly understood. Here, we report the function of a nuclear pre-mRNA 3'-end processing pathway in synapse and axon formation in C. elegans. In a genetic enhancer screen for synaptogenesis mutants, we identified a novel polyproline-rich protein, Synaptic defective enhancer-1 (SYDN-1). Loss of function of sydn-1 causes abnormal synapse and axon development, and displays striking synergistic interactions with several genes that regulate specific aspects of synapses. SYDN-1 is required in neurons and localizes to distinct regions of the nucleus. Through a genetic suppressor screen, we found that the neuronal defects of sydn-1 mutants are suppressed by loss of function in Polyadenylation factor subunit-2 (PFS-2), a conserved WD40-repeat protein that interacts with multiple subcomplexes of the pre-mRNA 3'-end processing machinery. PFS-2 partially colocalizes with SYDN-1, and SYDN-1 influences the nuclear abundance of PFS-2. Inactivation of several members of the nuclear 3'-end processing complex suppresses sydn-1 mutants. Furthermore, lack of sydn-1 can increase the activity of 3'-end processing. Our studies provide in vivo evidence for pre-mRNA 3'-end processing in synapse and axon development and identify SYDN-1 as a negative regulator of this cellular event in neurons.

The C. elegans peroxidasin PXN-2 is essential for embryonic morphogenesis and inhibits adult axon regeneration.

Peroxidasins form a highly conserved family of extracellular peroxidases of unknown cellular function. We identified the C. elegans peroxidasin PXN-2 in screens for mutants defective in embryonic morphogenesis. We find that PXN-2 is essential for specific stages of embryonic morphogenesis and muscle-epidermal attachment, and is also required postembryonically for basement membrane integrity. The peroxidase catalytic activity of PXN-2 is necessary for these developmental roles. pxn-2 mutants display aberrant ultrastructure of the extracellular matrix, suggesting a role in basement membrane consolidation. PXN-2 affects specific axon guidance choice points in the developing nervous system but is dispensable for maintenance of process positions. In adults, loss of pxn-2 function promotes regrowth of axons after injury, providing the first evidence that C. elegans extracellular matrix can play an inhibitory role in axon regeneration. Loss of function in the closely related C. elegans peroxidasin pxn-1 does not cause overt developmental defects. Unexpectedly, pxn-2 mutant phenotypes are suppressed by loss of function in pxn-1 and exacerbated by overexpression of wild-type pxn-1, indicating that PXN-1 and PXN-2 have antagonistic functions. These results demonstrate that peroxidasins play crucial roles in development and reveal a new role for peroxidasins as extracellular inhibitors of axonal regeneration.

Nanoscale patterning of collagens in C. elegans apical extracellular matrix.

Apical extracellular matrices (aECMs) are complex extracellular compartments that form important interfaces between animals and their environment. In the adult C. elegans cuticle, layers are connected by regularly spaced columnar structures known as struts. Defects in struts result in swelling of the fluid-filled medial cuticle layer ('blistering', Bli). Here we show that three cuticle collagens BLI-1, BLI-2, and BLI-6, play key roles in struts. BLI-1 and BLI-2 are essential for strut formation whereas activating mutations in BLI-6 disrupt strut formation. BLI-1, BLI-2, and BLI-6 precisely colocalize to arrays of puncta in the adult cuticle, corresponding to struts, initially deposited in diffuse stripes adjacent to cuticle furrows. They eventually exhibit tube-like morphology, with the basal ends of BLI-containing struts contact regularly spaced holes in the cuticle. Genetic interaction studies indicate that BLI strut patterning involves interactions with other cuticle components. Our results reveal strut formation as a tractable example of precise aECM patterning at the nanoscale.

Negative regulation of Caenorhabditis elegans epidermal damage responses by death-associated protein kinase.

Wounding of epidermal layers triggers multiple coordinated responses to damage. We show here that the Caenorhabditis elegans ortholog of the tumor suppressor death-associated protein kinase, dapk-1, acts as a previously undescribed negative regulator of barrier repair and innate immune responses to wounding. Loss of DAPK-1 function results in constitutive formation of scar-like structures in the cuticle, and up-regulation of innate immune responses to damage. Overexpression of DAPK-1 represses innate immune responses to needle wounding. Up-regulation of innate immune responses in dapk-1 requires the TIR-1/p38 signal transduction pathway; loss of function in this pathway synergizes with dapk-1 to drastically reduce adult lifespan. Our results reveal a previously undescribed function for the DAPK tumor suppressor family in regulation of epithelial damage responses.

Calcium and cyclic AMP promote axonal regeneration in Caenorhabditis elegans and require DLK-1 kinase.

Axons of adult Caenorhabditis elegans neurons undergo robust regenerative growth after laser axotomy. Here we show that axotomy of PLM sensory neurons triggers axonal calcium waves whose amplitude correlates with the extent of regeneration. Genetic elevation of Ca(2+) or cAMP accelerates formation of a growth cone from the injured axon. Elevated Ca(2+) or cAMP also facilitates apparent fusion of axonal fragments and promotes branching to postsynaptic targets. Conversely, inhibition of voltage-gated calcium channels or calcium release from internal stores reduces regenerative growth. We identify the fusogen EFF-1 as critical for axon fragment fusion and the basic leucine zipper domain (bZip) protein CREB (cAMP response element-binding protein) as a key effector for branching. The effects of elevated Ca(2+) or cAMP on regrowth require the MAPKKK (mitogen-activated protein kinase kinase kinase) DLK-1. Increased cAMP signaling can partly bypass the requirement for the bZip protein CEBP-1, a downstream factor of the DLK-1 kinase cascade. These findings reveal the relationship between Ca(2+)/cAMP signaling and the DLK-1 MAPK (mitogen-activated protein kinase) cascade in regeneration.

Distinct innate immune responses to infection and wounding in the C. elegans epidermis.

BACKGROUND: In many animals, the epidermis is in permanent contact with the environment and represents a first line of defense against pathogens and injury. Infection of the nematode Caenorhabditis elegans by the natural fungal pathogen Drechmeria coniospora induces the expression in the epidermis of antimicrobial peptide (AMP) genes such as nlp-29. Here, we tested the hypothesis that injury might also alter AMP gene expression and sought to characterize the mechanisms that regulate the innate immune response. RESULTS: Injury induces a wound-healing response in C. elegans that includes induction of nlp-29 in the epidermis. We find that a conserved p38-MAP kinase cascade is required in the epidermis for the response to both infection and wounding. Through a forward genetic screen, we isolated mutants that failed to induce nlp-29 expression after D. coniospora infection. We identify a kinase, NIPI-3, related to human Tribbles homolog 1, that is likely to act upstream of the MAPKK SEK-1. We find NIPI-3 is required only for nlp-29 induction after infection and not after wounding. CONCLUSIONS: Our results show that the C. elegans epidermis actively responds to wounding and infection via distinct pathways that converge on a conserved signaling cassette that controls the expression of the AMP gene nlp-29. A comparison between these results and MAP kinase signaling in yeast gives insights into the possible origin and evolution of innate immunity.

SYD-1, a presynaptic protein with PDZ, C2 and rhoGAP-like domains, specifies axon identity in C. elegans.

Axons are defined by the presence of presynaptic specializations at specific locations. We show here that loss-of-function mutations in the C. elegans gene syd-1 cause presynaptic specializations to form in the dendritic processes of GABA-expressing motor neurons during initial differentiation. At a later developmental stage, however, syd-1 is not required for the polarity respecification of a subset of these neurons. The SYD-1 protein contains PDZ, C2 and rho-GTPase activating protein (GAP)-like domains, and is localized to presynaptic terminals in mature neurons. A truncated SYD-1 that lacks the rhoGAP domain interferes with neurite outgrowth and guidance. Our data indicate that syd-1 may be involved in specifying axon identity during initial polarity acquisition.

High total organic carbon in surface waters of the northern Arabian Gulf: Implications for the oxygen minimum zone of the Arabian Sea.

Measurements of total organic carbon (TOC) for two years in Kuwaiti waters showed high TOC levels (101.0-318.4, mean 161.2 µM) with maximal concentrations occurring within the polluted Kuwait Bay and decreasing offshore, indicating substantial anthropogenic component. Analysis of winter-time data revealed a large increase in density over the past four decades due to decrease in Shatt Al-Arab runoff, implying that the dissolved/suspended organic matter in surface waters of the northern Gulf could be quickly injected into the Gulf Deep Water (GDW). Our measurements together with an analysis of previously collected/published data suggest that the recent summer-time declining trend in oxygen in the GDW might be related to eutrophication. Higher preformed TOC and lower preformed dissolved oxygen contents of the high-salinity water mass that flows out of the Gulf and ventilates the mesopelagic oxygen minimum zone (OMZ) of the Northwestern Indian Ocean may cause expansion/intensification of the regional OMZ.

Asynchronous Cholinergic Drive Correlates with Excitation-Inhibition Imbalance via a Neuronal Ca2+ Sensor Protein.

Excitation-inhibition imbalance in neural networks is widely linked to neurological and neuropsychiatric disorders. However, how genetic factors alter neuronal activity, leading to excitation-inhibition imbalance, remains unclear. Here, using the C. elegans locomotor circuit, we examine how altering neuronal activity for varying time periods affects synaptic release pattern and animal behavior. We show that while short-duration activation of excitatory cholinergic neurons elicits a reversible enhancement of presynaptic strength, persistent activation results to asynchronous and reduced cholinergic drive, inducing imbalance between endogenous excitation and inhibition. We find that the neuronal calcium sensor protein NCS-2 is required for asynchronous cholinergic release in an activity-dependent manner and dampens excitability of inhibitory neurons non-cell autonomously. The function of NCS-2 requires its Ca2+ binding and membrane association domains. These results reveal a synaptic mechanism implicating asynchronous release in regulation of excitation-inhibition balance.

Microtubule-dependent ribosome localization in C. elegans neurons.

Subcellular localization of ribosomes defines the location and capacity for protein synthesis. Methods for in vivo visualizing ribosomes in multicellular organisms are desirable in mechanistic investigations of the cell biology of ribosome dynamics. Here, we developed an approach using split GFP for tissue-specific visualization of ribosomes in Caenorhabditis elegans. Labeled ribosomes are detected as fluorescent puncta in the axons and synaptic terminals of specific neuron types, correlating with ribosome distribution at the ultrastructural level. We found that axonal ribosomes change localization during neuronal development and after axonal injury. By examining mutants affecting axonal trafficking and performing a forward genetic screen, we showed that the microtubule cytoskeleton and the JIP3 protein UNC-16 exert distinct effects on localization of axonal and somatic ribosomes. Our data demonstrate the utility of tissue-specific visualization of ribosomes in vivo, and provide insight into the mechanisms of active regulation of ribosome localization in neurons.

Dynamic microtubules drive circuit rewiring in the absence of neurite remodeling.

A striking neuronal connectivity change in C. elegans involves the coordinated elimination of existing synapses and formation of synapses at new locations, without altering neuronal morphology. Here, we investigate the tripartite interaction between dynamic microtubules (MTs), kinesin-1, and vesicular cargo during this synapse remodeling. We find that a reduction in the dynamic MT population in motor neuron axons, resulting from genetic interaction between loss of function in the conserved MAPKKK dlk-1 and an α-tubulin mutation, specifically blocks synapse remodeling. Using live imaging and pharmacological modulation of the MT cytoskeleton, we show that dynamic MTs are increased at the onset of remodeling and are critical for new synapse formation. DLK-1 acts during synapse remodeling, and its function involves MT catastrophe factors including kinesin-13/KLP-7 and spastin/SPAS-1. Through a forward genetic screen, we identify gain-of-function mutations in kinesin-1 that can compensate for reduced dynamic MTs to promote synaptic vesicle transport during remodeling. Our data provide in vivo evidence supporting the requirement of dynamic MTs for kinesin-1-dependent axonal transport and shed light on the role of the MT cytoskeleton in facilitating neural circuit plasticity.

Systematic analyses of rpm-1 suppressors reveal roles for ESS-2 in mRNA splicing in Caenorhabditis elegans.

The PHR (Pam/Highwire/RPM-1) family of ubiquitin E3 ligases plays conserved roles in axon patterning and synaptic development. Genetic modifier analysis has greatly aided the discovery of the signal transduction cascades regulated by these proteins. In Caenorhabditis elegans, loss of function in rpm-1 causes axon overgrowth and aberrant presynaptic morphology, yet the mutant animals exhibit little behavioral deficits. Strikingly, rpm-1 mutations strongly synergize with loss of function in the presynaptic active zone assembly factors, syd-1 and syd-2, resulting in severe locomotor deficits. Here, we provide ultrastructural evidence that double mutants, between rpm-1 and syd-1 or syd-2, dramatically impair synapse formation. Taking advantage of the synthetic locomotor defects to select for genetic suppressors, previous studies have identified the DLK-1 MAP kinase cascade negatively regulated by RPM-1. We now report a comprehensive analysis of a large number of suppressor mutations of this screen. Our results highlight the functional specificity of the DLK-1 cascade in synaptogenesis. We also identified two previously uncharacterized genes. One encodes a novel protein, SUPR-1, that acts cell autonomously to antagonize RPM-1. The other affects a conserved protein ESS-2, the homolog of human ES2 or DGCR14. Loss of function in ess-2 suppresses rpm-1 only in the presence of a dlk-1 splice acceptor mutation. We show that ESS-2 acts to promote accurate mRNA splicing when the splice site is compromised. The human DGCR14/ES2 resides in a deleted chromosomal region implicated in DiGeorge syndrome, and its mutation has shown high probability as a risk factor for schizophrenia. Our findings provide the first functional evidence that this family of proteins regulate mRNA splicing in a context-specific manner.

The microtubule minus-end-binding protein patronin/PTRN-1 is required for axon regeneration in C. elegans.

Precise regulation of microtubule (MT) dynamics is increasingly recognized as a critical determinant of axon regeneration. In contrast to developing neurons, mature axons exhibit noncentrosomal microtubule nucleation. The factors regulating noncentrosomal MT architecture in axon regeneration remain poorly understood. We report that PTRN-1, the C. elegans member of the Patronin/Nezha/calmodulin- and spectrin-associated protein (CAMSAP) family of microtubule minus-end-binding proteins, is critical for efficient axon regeneration in vivo. ptrn-1-null mutants display generally normal developmental axon outgrowth but significantly impaired regenerative regrowth after laser axotomy. Unexpectedly, mature axons in ptrn-1 mutants display elevated numbers of dynamic axonal MTs before and after injury, suggesting that PTRN-1 inhibits MT dynamics. The CKK domain of PTRN-1 is necessary and sufficient for its functions in axon regeneration and MT dynamics and appears to stabilize MTs independent of minus-end localization. Whereas in developing neurons, PTRN-1 inhibits activity of the DLK-1 mitogen-activated protein kinase (MAPK) cascade, we find that, in regeneration, PTRN-1 and DLK-1 function together to promote axonal regrowth.

Position of UNC-13 in the active zone regulates synaptic vesicle release probability and release kinetics.

The presynaptic active zone proteins UNC-13/Munc13s are essential for synaptic vesicle (SV) exocytosis by directly interacting with SV fusion apparatus. An open question is how their association with active zones, hence their position to Ca(2+) entry sites, regulates SV release. The N-termini of major UNC-13/Munc13 isoforms contain a non-calcium binding C2A domain that mediates protein homo- or hetero-meric interactions. Here, we show that the C2A domain of Caenorhabditis elegans UNC-13 regulates release probability of evoked release and its precise active zone localization. Kinetics analysis of SV release supports that the proximity of UNC-13 to Ca(2+) entry sites, mediated by the C2A-domain containing N-terminus, is critical for accelerating neurotransmitter release. Additionally, the C2A domain is specifically required for spontaneous release. These data reveal multiple roles of UNC-13 C2A domain, and suggest that spontaneous release and the fast phase of evoked release may involve a common pool of SVs at the active zone. DOI: http://dx.doi.org/10.7554/eLife.01180.001.

Liprin-α/SYD-2 determines the size of dense projections in presynaptic active zones in C. elegans.

Synaptic vesicle (SV) release is spatially and temporally regulated by a network of proteins that form the presynaptic active zone (AZ). The hallmark of most AZs is an electron-dense projection (DP) surrounded by SVs. Despite their importance for our understanding of triggered SV release, high-resolution analyses of DP structures are limited. Using electron microscopy, we show that DPs at Caenorhabditis elegans neuromuscular junctions (NMJs) were highly structured, composed of building units forming bays in which SVs are docked to the AZ membrane. Furthermore, larger ribbonlike DPs that were multimers of the NMJ building unit are found at synapses between inter- and motoneurons. We also demonstrate that DP size is determined by the activity of the AZ protein SYD-2/Liprin-α. Whereas loss of syd-2 function led to smaller DPs, syd-2 gain-of-function mutants displayed larger ribbonlike DPs through increased recruitment of ELKS-1/ELKS. Therefore, our data suggest that a main role of SYD-2/Liprin-α in synaptogenesis is to regulate the polymerization of DPs.

Kinesin-13 and tubulin posttranslational modifications regulate microtubule growth in axon regeneration.

The microtubule (MT) cytoskeleton of a mature axon is maintained in a stabilized steady state, yet after axonal injury it can be transformed into a dynamic structure capable of supporting axon regrowth. Using Caenorhabditis elegans mechanosensory axons and in vivo imaging, we find that, in mature axons, the growth of MTs is restricted in the steady state by the depolymerizing kinesin-13 family member KLP-7. After axon injury, we observe a two-phase process of MT growth upregulation. First, the number of growing MTs increases at the injury site, concomitant with local downregulation of KLP-7. A second phase of persistent MT growth requires the cytosolic carboxypeptidase CCPP-6, which promotes Δ2 modification of α-tubulin. Both phases of MT growth are coordinated by the DLK-1 MAP kinase cascade. Our results define how the stable MT cytoskeleton of a mature neuron is converted into the dynamically growing MT cytoskeleton of a regrowing axon.

Motor neuron synapse and axon defects in a C. elegans alpha-tubulin mutant.

Regulation of microtubule dynamics underlies many fundamental cellular mechanisms including cell division, cell motility, and transport. In neurons, microtubules play key roles in cell migration, axon outgrowth, control of axon and synapse growth, and the regulated transport of vesicles and structural components of synapses. Loss of synapse and axon integrity and disruption of axon transport characterize many neurodegenerative diseases. Recently, mutations that specifically alter the assembly or stability of microtubules have been found to directly cause neurodevelopmental defects or neurodegeneration in vertebrates. We report here the characterization of a missense mutation in the C-terminal domain of C. elegans alpha-tubulin, tba-1(ju89), that disrupts motor neuron synapse and axon development. Mutant ju89 animals exhibit reduction in the number and size of neuromuscular synapses, altered locomotion, and defects in axon extension. Although null mutations of tba-1 show a nearly wild-type pattern, similar axon outgrowth defects were observed in animals lacking the beta-tubulin TBB-2. Genetic analysis reveals that tba-1(ju89) affects synapse development independent of its role in axon outgrowth. tba-1(ju89) is an altered function allele that most likely perturbs interactions between TBA-1 and specific microtubule-associated proteins that control microtubule dynamics and transport of components needed for synapse and axon growth.