Schaan Profes, Marcos et al. (2021) International Worm Meeting "How do neurexins promote presynaptic development?"

Kurshan, Peri, Schaan Profes, Marcos

[International Worm Meeting, 2021]

Synapse development is critical for the formation of neuronal networks while defects in synapse formation lead to neurodevelopmental disorders. Cell adhesion molecule signaling plays a prominent role in synapse formation, and strong evidence has emerged linking dysfunction of the cell adhesion molecule neurexin to autism spectrum disorders and intellectual disability. However, the precise role of this molecule in regulating synapse formation remains elusive. In humans, three neurexin genes are expressed as thousands of splice isoforms, all of which share a conserved intracellular domain. Understanding the intracellular signaling pathway mediated by neurexin may lead to the development of therapeutics for a multitude of neurexin-associated disorders. C. elegans has long been established as a powerful tool for gene discovery. To elucidate which proteins interact with neurexin's intracellular domain (ICD) we are employing a recently developed enzyme-catalyzed proximity labeling method called TurboID. In TurboID, a protein of interest is tagged with a promiscuous labeling enzyme - the E. coli biotin ligase BirA - which biotinylates neighboring proteins located within a few nanometer radius. Biotinylated proteins are pulled down using streptavidin. To determine the appropriate position for insertion of BirA within neurexin's ICD while not interfering with neurexin's function, we engineered transgenes with various BirA locations. Transgenes carrying BirA inserted just before the C-terminal PDZ binding motif fully rescued the synaptic assembly defects observed in neurexin null mutants, so we chose this location for endogenous insertion via CRISPR/Cas9. The endogenously-tagged BirA strain was then validated using our previously developed presynaptic assembly markers and showed no synaptic defects when compared to wild-type animals. Streptavidin pull-downs followed by mass spectrometry were performed using our endogenously-tagged neurexin-BirA strain and 70 potential neurexin ICD interactors were identified when compared to controls (cytosolic BirA and wild type strains). Of those, we are following up on 21 hits that, by gene ontology analysis, include cytoskeletal-related, endo/exocytitc, metabolic and adhesion processes. This approach will reveal the signaling pathway downstream of neurexin with the potential of identifying therapeutic targets widely applicable to various neurexin-associated disorders.

Schaan Profes, Marcos et al. (2021) International Worm Meeting "Developing a single-synapse functional imaging assay in C. elegans"

Schaan Profes, Marcos, Tiroumalechetty, Araven, Kurshan, Peri

[International Worm Meeting, 2021]

Individual synapses in a neuronal circuit-or even within a single neuron-often exhibit dramatically different levels of activity from one another. To better understand the mechanisms regulating functional heterogeneity within neuronal circuits we are developing a single-synapse functional imaging assay in C. elegans. A membrane-bound version of the genetically encoded calcium indicator GCaMP5 was targeted postsynaptically at neuromuscular junctions, revealing fluctuating fluorescent transients opposed to individual presynaptic release sites and dependent on the synaptic vesicle release protein UNC-13. Spontaneous calcium transients are analyzed using a custom and automated Matlab suite we have developed. Our analysis pipeline enables us to identify waves of synaptic transmission propagating along an axon via en passant synapses. Combining this assay with cell-specific presynaptic markers will enable us to understand how synaptic activity differs between synapses in the same neuron and between neighboring motor neurons of different classes. In addition to spontaneous activity, we will use optogenetic tools to assess evoked synaptic activity and plasticity. Finally, we will assess how synaptic heterogeneity or single-synapse functional properties are affected by mutations in presynaptic proteins including neurexin, a synaptic cell-adhesion molecule associated with functional maturation of synapses and implicated in neurodevelopmental disorders such as Autism.

Frankel, Elisa et al. (2021) International Worm Meeting "SYG-2/nephrin mediates incorporation of new synapses into preexisting circuits"

Dong, Yongming, Frankel, Elisa, Bai, Jihong, Kurshan, Peri

[International Worm Meeting, 2021]

Genetic insults that disrupt synaptogenic proteins have been well established as risk factors for an array of heritable neurodevelopmental and neuropsychiatric disorders, but the mechanisms by which these mutations perturb synapse assembly may differ at various developmental stages. Leveraging the stereotypical neuronal circuitry, genetic tractability, and optical accessibility of the nematode C. elegans, we are investigating the molecular mechanisms promoting the incorporation of new synapses into preexisting circuits. A screen in the C. elegans DA9 motor neuron revealed that the nephrin family neuronal cell adhesion molecule SYG-2 cell-autonomously regulates the number, size, and spacing of excitatory motor neuron synapses at late developmental stages. We found that SYG-2::GFP localizes to the entire presynaptic domain of DA9, rather than specifically at synapses like other canonical neuronal cell adhesion molecules (such as neurexin), and additionally that SYG-2 acts in the same pathway as NCK-1, a protein involved in actin cytoskeleton remodeling. We hypothesize that SYG-2 functions to regulate the addition of new synapses into preexisting circuits by modulating local cytoskeletal elements through interaction with NCK-1, and we are interrogating this hypothesis using structure/function studies to identify the domain(s) of syg-2/nephrin that regulate its cell-autonomous functions and interact with cytoskeletal remodeling pathways. An integrated understanding of synaptogenesis at distinct stages of neural circuit development may shed light on how molecular disruptions to synapse formation manifest as neurodevelopmental disorders.

Schlesinger, E. et al. (2019) International Worm Meeting "The Effects of DAF-16/FOXO Translocation on Quiescent Peri-Hatching Arrest in Caenorhabditis elegans."

Wightman, B., Mesrobian, A., Liberatore, K., Schlesinger, E.

[International Worm Meeting, 2019]

The sole insulin receptor in C. elegans, daf-2, regulates aging and the formation of a non-feeding, stress-resistant diapause state known as dauer via a signaling pathway that results in the phosphorylation and resultant cytoplasmic sequestering of the transcription factor daf-16/FOXO. We found that pairing daf-2(e1370) with a mutation in the nuclear hormone receptor fax-1, which regulates interneuron identity and axon pathfinding, results in a unique quiescent peri-hatching arrest that can be reversed by waking larvae with an aversive stimulus in the form of blue light. A similar phenotype is observed in a daf-18; fax-1 double mutant, indicating that the peri-hatching arrest depends on the canonical PTEN signaling pathway. We propose that the sleep-like arrest phenotype may be caused by interaction between the daf-2 mediated insulin growth factor pathway and an interneuron developmentally regulated by fax-1. daf-2 exhibits complex epistasis relationships with different alleles. The dauer formation phenotype of class I alleles such as daf-2(m41) is suppressed by a mutation in daf-12, a nuclear hormone receptor that regulates dauer formation in response to steroid hormone signaling, while that of class II alleles such as daf-2(e1370) is enhanced. Unlike class II alleles, class I daf-2 alleles do not cause a peri-hatching arrest in combination with fax-1 mutations. Interestingly, daf-12(m20) suppresses the peri-hatching arrest of fax-1(gm83);daf-2(e1370) mutants, even though it does not suppress dauer arrest. This observation suggests that the peri-hatching arousal pathway works differently than the dauer pathway. Dauer arrest promoted by daf-2 mutations depends on daf-16/FOXO. The peri-hatching arrest promoted by daf-2;fax-1 double mutants does so also. We are investigating whether peri-hatching arrest depends on translocation of DAF-16/FOXO into the nucleus.

Frankel, Elisa et al. (2021) International Worm Meeting "Neurexin clustering at synapses is mediated by active zone scaffold intrinsically disordered domains"

Tiroumalechetty, Araven, Henry, Parise, Kurshan, Peri, Rogow, Jackson, Frankel, Elisa

[International Worm Meeting, 2021]

Synaptic cell-adhesion molecules (sCAMs) are thought to mediate the formation of neuronal circuits by recognizing appropriate trans-synaptic partners and initiating the assembly of pre- and post-synaptic specializations. Of the sCAMs, neurexins are among the most extensively studied, and all neurexin family members have been identified as risk factors for autism and other neurodevelopmental disorders. The human genome contains three neurexin genes which, through alternative splicing, encode ~4000 long (alpha), medium (beta) and short (gamma) isoforms that differ in their extracellular domain but retain an identical intracellular domain. The C. elegans genome contains a single neurexin gene nrx-1, which is expressed only as several long (alpha) and short (gamma) isoforms that also share a common intracellular domain. To evaluate whether nrx-1 isoforms are differentially expressed across the C. elegans nervous system, we generated promoter reporters for the short (gamma) and long (alpha) isoforms, and found differential expression patterns. We are now using the NeuroPal tool to identify the specific neurons in which these isoforms are expressed. In addition, we have generated long and short isoform-specific knockouts of endogenously-tagged nrx-1 to reveal the contribution of each isoform to overall synaptic expression. We have previously shown that a short (gamma) isoform of NRX-1 that doesn't include any canonical extracellular binding domains nonetheless localizes to presynaptic active zones and regulates presynaptic maturation and stability. We now show that expression of the intracellular domain alone, targeted to the membrane with a myristoylation sequence, can rescue the nrx-1 null phenotype (although a cytosolic version does not), indicating that the membrane-bound NRX-1 intracellular domain is sufficient for mediating its presynaptic assembly functions. To identify mediators of NRX-1 localization at synapses, we performed a candidate screen of potential intracellular interactors using endogenously-tagged NRX-1 and identified the synaptic vesicle kinesin UNC-104, as well as active zone scaffold molecules SYD-1 and SYD-2. A deletion of the intrinsically disordered domain of SYD-2, shown previously to mediate its ability to undergo phase separation and thereby recruit additional active zone molecules, affected NRX-1 localization to the same degree as syd-2 null mutants, implicating phase separation as a potential mechanism for NRX-1 recruitment to synapses.

Zuluaga-Forero, Maximiliano et al. (2021) International Worm Meeting "Novel patient-derived mutation in the presynaptic calcium channel UNC-2 reduces synaptic expression yet increases presynaptic release"

Kurshan, Peri T., Dong, Yongming, Bai, Jihong, Zuluaga-Forero, Maximiliano

[International Worm Meeting, 2021]

Channelopathies cause a wide variety of disorders, but understanding how specific mutations lead to disease has been complicated by gene redundancy and compensatory effects in various classical and complex vertebrate models. We use C. elegans to evaluate the functional and behavioral effect of a rare and novel human patient-derived point mutation (D1634N) in the P/Q-type voltage-gated calcium channel (CACNA1). Mutations in this channel are typically associated with autosomal dominant neurological disorders like familial hemiplegic migraine type 1 (FHM1), episodic ataxia type 2 (EA-2), and Spinocerebellar ataxia type 6 (SCA6); however, patients with the D1634N mutation have not been classified into any of these disorders, suggesting a novel mechanism of action. The worm has only one ortholog of the channel (UNC-2) expressed in its nervous system and shares high homology in the region of the single point mutation D1634N. Our CRISPR-generated D1634N worms show decreased channel expression at synapses using an endogenously-tagged protein. Surprisingly, acetylcholine release (as assessed by aldicarb sensitivity), miniature EPSC frequency, and behavioral readouts of hyperactivity are all increased, suggesting that this point mutation may affect channel voltage sensitivity and/or sub-synaptic localization at presynapses. Additional interactions with other presynaptic proteins and calcium channel auxiliary subunits are being evaluated to address the impact of this mutation on presynaptic morphology and synaptic function.

San Miguel, Adriana et al. (2013) International Worm Meeting "Phenotypic profiling of synaptic sites for subtle mutant identification in automated genetic screens."

Crane, Matthew, Lu, Hang, Shen, Kang, Kurshan, Peri, San Miguel, Adriana

[International Worm Meeting, 2013]

Many neurological diseases, caused by defects in neurotransmission, are suspected to show no obvious morphological alterations in neuronal patterning. Uncovering genes responsible for these disorders by identifying mutants with subtle changes in morphology of micron-sized synapses is a very challenging task that necessitates high-resolution and quantitative phenotyping, making genetic screening virtually impossible. In this work we exploit an integrated approach to perform automated genetic screens in C. elegans that enables the isolation of mutants with extremely subtle phenotypic differences in synaptic morphology. We take advantage of microfluidic devices for easy worm handling, paired with automated control of flow, image analysis, and decision-making. Computer vision algorithms allow the unsupervised detection of fluorescently labeled micron-sized synaptic puncta in an unbiased and quantitative manner. Phenotypic profiling of synaptic sites is performed by quantification of a large number of descriptors relevant for synapse morphology, such as synapse size and intensity. In order to take into account the heterogeneity present from isogenic populations, statistical analyses are performed based on phenotypic profiling of whole populations. Logistic regression models allow discerning between populations of true mutants and wild type animals, and suggest the most relevant features that differentiate them. The phenotypic differences of the mutants found in this work range from smaller, dimmer synaptic puncta, altered number, density and interpunctal distance, all extremely difficult to discern by eye. Performing full profile analysis of the found mutants, we are able to suggest putative genetic pathways in which the newly found alleles belong by analyzing their relationships with a representative collection of mutants of known pathways. Overall, the approach shown here provides a practical, feasible technique to perform extraordinarily difficult genetic screens in an unsupervised and unbiased manner and enables the isolation of mutants with extremely subtle phenotypic differences.

Wightman, Bruce et al. (2019) International Worm Meeting "Insulin-Dependent Quiescence and Arrest at Hatching."

Wightman, Bruce, He, Zhengying

[International Worm Meeting, 2019]

The fax-1 nuclear hormone receptor and unc-42 homeobox gene control interneuron identities in C. elegans. Both transcription factors function in specifying the identities of an overlapping subset of nematode interneurons including AVA, AVE, and AVK. Both fax-1 and unc-42 mutations cause a novel peri-hatching arrest in combination with daf-2 insulin receptor mutations. Mutations in both fax-1 and unc-42 also enhance other aspects of insulin pathway function, including dauer formation and adult arrest, but not longevity, suggesting that disrupting the differentiation of specific interneurons leads to a reduction in insulin signaling. Peri-hatching arrest can be reversed by a mutation in the daf-16 forkhead transcription factor, which functions downstream of daf-2, but not by mutations in the parallel TGF? pathway. Arrest can also be suppressed by mutations in the daf-12 nuclear receptor, indicating that the canonical insulin and steroid pathways that regulate dauer formation also promote arousal and developmental progression at hatching. Arrested fax-1;daf-2 and unc-42;daf-2 embryos typically display normal L1 morphology, but often remain coiled in a broken eggshell in a state of quiescence with weak or absent pharyngeal pumping. Arrested embryos can be prompted to vigorous movement by stimulation with blue light. Temperature-shift experiments suggest that embryogenesis, not L1 feeding after hatching, is the critical stage for quiescence and arrest. This phenotype is similar to that observed when embryos are subjected to osmotic stress. The ssu-1 sulfotransferase gene is expressed in the ASJ sensory neurons and is required for peri-hatching arrest in response to NaCl, which is also dependent on insulin signaling (Burton et al., 2018). The peri-hatching arrest of both fax-1;daf-2 and unc-42;daf-2 double mutants is suppressed by a mutation in ssu-1. This result places the interneuron peri-hatching arousal function downstream of the osmotic stress signal and a putative steroid signal produced by the ASJ interneurons. Several models are consistent with the combined action of a steroid signaling pathway and neuroendocrine functions of insulin signaling in promoting arousal and developmental progression at hatching.

Sun, Chun-Ling et al. (2009) International Worm Meeting "Genetic characterization of the CED-4 translocation event during apoptosis."

Friedman, Josh, Wang, Xiaochen, Lai, Huey-Jen, Parrish, Jay, Xue, Ding, Sun, Chun-Ling

[International Worm Meeting, 2009]

Apoptosis plays an essential role in maintaining tissue homeostasis and animal development. In C. elegans, the BH3-only cell death initiator EGL-1 induces cell death by binding to CED-9 and causing release of the CED-4 dimer from the CED-4/CED-9 complex tethered on the surface of mitochondria (1, 2). CED-4 dimers then further oligomerize to result in activation of the CED-3 zymogen. Intriguingly, upon release from the surface of mitochondria, CED-4 translocates to the peri-nuclear membrane but the role of such translocation in regulating cell death activation is not understood (1). We have carried out a CED-4::GFP based genetic screen to look for mutations that block translocation of CED-4 to the peri-nuclear membrane and have isolated four mutations that result in mis-localization of CED-4 (mic). These four mic mutations define three new genes, mic-1(sm158, sm160), mic-2(sm161), and mic-3(sm162). Phenotypic analysis reveals that mic-1 mutations cause stronger Mic phenotype than mic-2 and mic-3 mutations. Although these mic mutants alone do not display an obvious cell death defect, they can block cell death in sensitized genetic backgrounds. Our results suggest that translocation of CED-4 from mitochondria to the peri-nuclear membrane may play an important regulatory role in cell death activation. We have recently cloned the mic-1 gene and are in the process of cloning mic-2 and mic-3. Further molecular and biochemical characterization of MIC proteins should reveal how CED-4 translocates to the peri-nuclear membrane and what its role is in cell death activation in C. elegans. 1. Chen, F. et al., Translocation of C. elegans CED-4 to nuclear membranes during programmed cell death. Science 287 (5457), 1485-1489 (2000). 2. Yan, N. et al., Structure of the CED-4-CED-9 complex provides insights into programmed cell death in Caenorhabditis elegans. Nature 437 (7060), 831-837 (2005).

Haspel, Gal et al. (2011) International Worm Meeting "A new connectivity model for the locomotion network."

O'Donovan, Michael J., Haspel, Gal

[International Worm Meeting, 2011]

Seventy five motorneurons of eight classes innervate the body musculature that propels Caenorhabditis elegans with dorsoventral undulations. These motorneurons are interconnected by synapses and gap junctions to create a motorneuronal network. The nervous system of C. elegans has been reconstructed from electron micrographs and provides a connectivity data set that is unavailable for any other animal model. However, the dataset is incomplete. The ventral and dorsal nerve cords of a single nematode were reconstructed halfway along the body and the posterior data is missing, leaving 21 of 75 motorneurons of the locomotor network with partial or no connectivity data. Using a new framework for network analysis, the peri-motor space, we identified rules of connectivity that allowed us to approximate the missing data by extrapolation. We mapped the motorneurons in peri-motor space instead of the formerly used mapping coordinates that were either arbitrary or derived from connectivity. In the new peri-motor frame of reference, each motorneuron is located according to the muscle cells it innervates. In this framework, a pattern of iterative connections emerges which includes most (0.90) of the connections. We identified a repeating unit consisting of 12 motorneurons and 12 muscle cells. The cell bodies of the motorneurons of such a unit are not necessarily anatomical neighbors and there is no obvious anatomical segmentation. A connectivity model, composed of six repeating units, is a description of the network that is both simplified (omitting non-iterative connections and modular) and more complete (includes the posterior part) than the original data set. The peri-motor framework of observed connectivity and the segmented connectivity model give insights and advance the study of the neuronal infrastructure underlying locomotion in C. elegans. We are using the model of the motorneuronal network to give context to the recorded activity of motorneurons during locomotion. We expressed a calcium sensor in subsets of motorneurons to record their activity and correlate it with the locomotor behavior. We found that some classes of motorneurons are dedicated to either forward or backward locomotion, forming two overlapping motorneuronal networks. A neuronal network comprised of direction-specific classes of motorneurons, might be an ancestral form of locomotor control to which dedicated and multifunctional interneurons were subsequently added.