Background: Understanding gene function and genetic relationships is fundamental to our efforts to better understand biological systems. Previous studies systematically describing genetic interactions on a global scale have either focused on core biological processes in protozoans or surveyed catastrophic interactions in metazoans. Here, we describe a reliable high-throughput approach capable of revealing both weak and strong genetic interactions in the nematode Caenorhabditis elegans.

Summary The ability of injured axons to regenerate declines with age yet the mechanisms that regulate axon regeneration in response to age are not known. Here we show that axon regeneration in aging C. elegans motor neurons is inhibited by the conserved insulin/IGF1 receptor DAF-2. DAF-2’s function in regeneration is mediated by intrinsic neuronal activity of the forkhead transcription factor DAF-16/FOXO. DAF-16 regulates regeneration independently of lifespan, indicating that neuronal aging is an intrinsic, neuron specific, and genetically regulated process. In addition, we found that daf-18/PTEN inhibits regeneration independently of age and FOXO signaling, via the TOR pathway. Finally, DLK-1, a conserved regulator of regeneration, is downregulated by insulin/IGF1 signaling, bound by DAF-16 in neurons, and is required for both DAF-16- and DAF-18-mediated regeneration. Together, our data establish that insulin signaling specifically inhibits regeneration in aging adult neurons, and that this mechanism is independent of PTEN and TOR.

The postsynaptic membrane of the embryonic neuromuscular junction undergoes a dramatic expansion during later development to facilitate the depolarization of larger muscles. In C. elegans, the postsynaptic membrane resides at the termini of plasma membrane extensions called muscle arms. Membrane extension to the motor axons during larval development doubles the number of muscle arms, making them a tractable model to investigate both postsynaptic membrane expansion and guided membrane extension. To identify genes required for muscle arm extension, we performed a forward screen for mutants with fewer muscle arms. We isolated 23 mutations in 14 genes, including unc-40/Dcc, which encodes a transmembrane receptor that guides the migration of cells and extending axons in response to the secreted UNC-6/Netrin spatial cue. We discovered that UNC-40 is enriched at muscle arm termini and functions cell-autonomously to direct arm extension to the motor axons. Surprisingly, UNC-6 is dispensable for muscle arm extension, suggesting that UNC-40 relies on other spatial cues to direct arm extension. We provide the first evidence that the guanine-nucleotide exchange factor UNC-73/Trio, members of the WAVE actin-polymerization complex, and a homolog of the focal adhesion complex can function downstream of UNC-40 to direct membrane extension. Our work is the first to define a pathway for directed muscle membrane extension and illustrates that axon guidance components can play key roles in postsynaptic membrane expansion.

The body muscles of Caenorhabditis elegans extend plasma membrane extensions called muscle arms to the midline motor axons to form the postsynaptic membrane of the neuromuscular junction. Through a screen for muscle arm development defective (Madd) mutants, we previously discovered that the UNC-40/DCC guidance receptor directs muscle arm extension through the Rho-GEF UNC-73. Here, we describe a gene identified through our mutant screen called madd-2, and show that it functions in an UNC-40 pathway. MADD-2 is a C1-TRIM protein and a homolog of human MID1, mutations in which cause Opitz Syndrome. We demonstrate that MADD-2 functions cell autonomously to direct muscle and axon extensions to the ventral midline of worms. Our results suggest that MADD-2 may enhance UNC-40 pathway activity by facilitating an interaction between UNC-40 and UNC-73. The analogous phenotypes that result from MADD-2 and MID1 mutations suggest that C1-TRIM proteins may have a conserved biological role in midline-oriented developmental events.

Sterile alpha and toll/interleukin receptor (TIR) motif-containing protein 1 (SARM1) is a neuronally expressed NAD+ glycohydrolase whose activity is increased in response to stress. NAD+ depletion triggers axonal degeneration, which is a characteristic feature of neurological diseases. Notably, loss of SARM1 is protective in murine models of peripheral neuropathy and traumatic brain injury. Herein, we report that citrate induces a phase transition that enhances SARM1 activity by ~2000-fold. This phase transition can be disrupted by mutating a residue involved in multimerization, G601P. This mutation also disrupts puncta formation in cells. We further show that citrate induces axonal degeneration in C. elegans that is dependent on the C. elegans orthologue of SARM1 (TIR-1). Notably, citrate induces the formation of larger puncta indicating that TIR-1/SARM1 multimerization is essential for degeneration in vivo. These findings provide critical insights into SARM1 biology with important implications for the discovery of novel SARM1-targeted therapeutics.