SUMMARY Sensory dendrites depend on cues from their environment to pattern their growth and direct them toward their correct target tissues. Yet, little is known about dendrite-substrate interactions during dendrite morphogenesis. Here, we describe MNR-1/menorin, which is part of the conserved Fam151 family of proteins and is expressed in the skin to control the elaboration of “menorah”-like dendrites of mechanosensory neurons in Caenorhabditis elegans. We provide biochemical and genetic evidence that MNR-1 acts as a contact-dependent or short-range cue in concert with the neural cell adhesion molecule SAX-7/L1CAM in the skin and through the neuronal leucine-rich repeat transmembrane receptor DMA-1 on sensory dendrites. Our data describe an unknown pathway that provides spatial information from the skin substrate to pattern sensory dendrite development nonautonomously.

Inhibitory GABAergic transmission is required for proper circuit function in the nervous system. However, our understanding of molecular mechanisms that preferentially influence GABAergic transmission, particularly presynaptic mechanisms, remains limited. We previously reported that the ubiquitin ligase EEL-1 preferentially regulates GABAergic presynaptic transmission. To further explore how EEL-1 functions, here we performed affinity purification proteomics using Caenorhabditis elegans and identified the O-GlcNAc transferase OGT-1 as an EEL-1 binding protein. This observation was intriguing, as we know little about how OGT-1 affects neuron function. Using C. elegans biochemistry, we confirmed that the OGT-1/EEL-1 complex forms in neurons in vivo and showed that the human orthologs, OGT and HUWE1, also bind in cell culture. We observed that, like EEL-1, OGT-1 is expressed in GABAergic motor neurons, localizes to GABAergic presynaptic terminals, and functions cell-autonomously to regulate GABA neuron function. Results with catalytically inactive point mutants indicated that OGT-1 glycosyltransferase activity is dispensable for GABA neuron function. Consistent with OGT-1 and EEL-1 forming a complex, genetic results using automated, behavioral pharmacology assays showed that ogt-1 and eel-1 act in parallel to regulate GABA neuron function. These findings demonstrate that OGT-1 and EEL-1 form a conserved signaling complex and function together to affect GABA neuron function. GABA neurons are a critical component of nervous systems across the animal kingdom from mammals (1, 2) to simple invertebrates, such as Caenorhabditis elegans (3, 4). They provide essential inhibitory activity within neural circuits. In humans, various dysfunctions in GABA neurons and the imbalance between excitatory and inhibitory neurotransmission contribute to neurodevelopmental disorders (5, 6). Thus, understanding how GABA neuron function is regulated is critical for our understanding of nervous system function and disease. Much remains unknown about molecular mechanisms that preferentially affect GABAergic transmission. Core presynaptic machinery, such as synaptotagmin, the SNARE complex, and active zone proteins, influence both glutamatergic and GABAergic transmission (7, 8). A few post-synaptic regulators that preferentially or specifically affect GABAergic transmission are known, including Gephyrin, Neuroligin2, Slitrk3, and GARHLs (9-13). In mammals, less is known about presynaptic GABA-specific regulators, but some proteins, such as synapsins, can differentially impact inhibitory transmission compared with excitatory transmission (14, 15). In C. elegans, core presynaptic components play conserved roles in neurotransmission in the motor circuit, a model circuit with balanced excitatory cholinergic and inhibitory GABAergic neuron function (4, 16). Like mammals, relatively few proteins are known that preferentially regulate presynaptic GABA function in C. elegans. Nonetheless, the worm motor circuit has proven valuable for identifying mol...

Autophagy is an intracellular catabolic process prominent in starvation, aging and disease. Neuronal autophagy is particularly important, as it affects the development and function of the nervous system, and is heavily implicated in neurodegenerative disease. Nonetheless, how autophagy is regulated in neurons remains poorly understood. Using an unbiased proteomics approach, we demonstrate that the primary initiator of autophagy, the UNC-51/ULK kinase, is negatively regulated by the ubiquitin ligase RPM-1. RPM-1 ubiquitin ligase activity restricts UNC-51 and autophagosome formation within specific axonal compartments, and exerts effects broadly across the nervous system. By restraining UNC-51 activity, RPM-1 inhibits autophagosome formation to affect axon termination, synapse maintenance and behavioral habituation. These results demonstrate how UNC-51 and autophagy are regulated subcellularly in axons, and unveils a mechanism for restricting initiation of autophagy across the nervous system. Our findings have important implications beyond nervous system development, given growing links between altered autophagy regulation and neurodegenerative diseases.

Understanding animal behavior and development requires visualization and analysis of their synaptic connectivity, but existing methods are laborious or may not depend on trans-synaptic interactions. Here we describe a transgenic approach for in vivo labeling of specific connections in Caenorhabditis elegans, which we term iBLINC. The method is based on BLINC (Biotin Labeling of INtercellular Contacts) and involves trans-synaptic enzymatic transfer of biotin by the Escherichia coli biotin ligase BirA onto an acceptor peptide. A BirA fusion with the presynaptic cell adhesion molecule NRX-1/neurexin is expressed presynaptically, whereas a fusion between the acceptor peptide and the postsynaptic protein NLG-1/neuroligin is expressed postsynaptically. The biotinylated acceptor peptide::NLG-1/neuroligin fusion is detected by a monomeric streptavidin::fluorescent protein fusion transgenically secreted into the extracellular space. Physical contact between neurons is insufficient to create a fluorescent signal, suggesting that synapse formation is required. The labeling approach appears to capture the directionality of synaptic connections, and quantitative analyses of synapse patterns display excellent concordance with electron micrograph reconstructions. Experiments using photoconvertible fluorescent proteins suggest that the method can be utilized for studies of protein dynamics at the synapse. Applying this technique, we find connectivity patterns of defined connections to vary across a population of wild-type animals. In aging animals, specific segments of synaptic connections are more susceptible to decline than others, consistent with dedicated mechanisms of synaptic maintenance. Collectively, we have developed an enzyme-based, trans-synaptic labeling method that allows high-resolution analyses of synaptic connectivity as well as protein dynamics at specific synapses of live animals. KEYWORDS biotin; circuit; connectome; labeling; synapse U NDERSTANDING animal behavior requires the determination and analysis of their precise neural connectivity, i.e., their connectome. Historically, this has been accomplished through reconstruction of serial sections of electron micrographs (White et al. 1986;Jarrell et al. 2012). Pioneered in the nematode Caenorhabditis elegans with its defined and invariant nervous system, partial circuits from Drosophila and the mouse retina have now also been reconstructed (Helmstaedter et al. 2013;Takemura et al. 2013). However, both the experimental effort and the static nature of the connectome at the time of analysis render investigations into the variability of synaptic connections between individuals, during development, or in different genotypes laborious at best. Live-imaging techniques to visualize synaptic connectivity have been developed such as GFP Reconstitution Across Synaptic Partners (GRASP) in C. elegans and, more recently, Synaptic Tagging with Recombination (STaR) in flies (Feinberg et al. 2008;Chen et al. 2014). GRASP takes advantage of the strong molecular intera...