Ubiquitin ligase and signalling hub MYCBP2 is required for efficient EPHB2 tyrosine kinase receptor function.

Eph receptor tyrosine kinases participate in a variety of normal and pathogenic processes during development and throughout adulthood. This versatility is likely facilitated by the ability of Eph receptors to signal through diverse cellular signalling pathways: primarily by controlling cytoskeletal dynamics, but also by regulating cellular growth, proliferation, and survival. Despite many proteins linked to these signalling pathways interacting with Eph receptors, the specific mechanisms behind such links and their coordination remain to be elucidated. In a proteomics screen for novel EPHB2 multi-effector proteins, we identified human MYC binding protein 2 (MYCBP2 or PAM or Phr1). MYCBP2 is a large signalling hub involved in diverse processes such as neuronal connectivity, synaptic growth, cell division, neuronal survival, and protein ubiquitination. Our biochemical experiments demonstrate that the formation of a complex containing EPHB2 and MYCBP2 is facilitated by FBXO45, a protein known to select substrates for MYCBP2 ubiquitin ligase activity. Formation of the MYCBP2-EPHB2 complex does not require EPHB2 tyrosine kinase activity and is destabilised by binding of ephrin-B ligands, suggesting that the MYCBP2-EPHB2 association is a prelude to EPHB2 signalling. Paradoxically, the loss of MYCBP2 results in increased ubiquitination of EPHB2 and a decrease of its protein levels suggesting that MYCBP2 stabilises EPHB2. Commensurate with this effect, our cellular experiments reveal that MYCBP2 is essential for efficient EPHB2 signalling responses in cell lines and primary neurons. Finally, our genetic studies in Caenorhabditis elegans provide in vivo evidence that the ephrin receptor VAB-1 displays genetic interactions with known MYCBP2 binding proteins. Together, our results align with the similarity of neurodevelopmental phenotypes caused by MYCBP2 and EPHB2 loss of function, and couple EPHB2 to a signalling effector that controls diverse cellular functions.

Axon development is regulated at genetic and proteomic interfaces between the integrin adhesome and the RPM-1 ubiquitin ligase signaling hub.

Integrin signaling plays important roles in development and disease. An adhesion signaling network called the integrin adhesome has been principally defined using bioinformatics and proteomics. To date, the adhesome has not been studied using integrated proteomic and genetic approaches. Here, proteomic studies in C. elegans identified physical associations between the RPM-1 ubiquitin ligase signaling hub and numerous adhesome components including Talin, Kindlin and beta-integrin. C. elegans RPM-1 is orthologous to human MYCBP2, a prominent player in nervous system development associated with a neurodevelopmental disorder. Using neuron-specific, CRISPR loss-of-function strategies, we show that core adhesome components affect axon development and interact genetically with RPM-1. Mechanistically, Talin opposes RPM-1 in a functional 'tug-of-war' on growth cones that is required for accurate axon termination. Thus, our findings orthogonally validate the adhesome via multi-component genetic and physical interfaces with a key neuronal signaling hub and identify new links between the adhesome and brain disorders.

Ubiquitin ligase and signalling hub MYCBP2 is required for efficient EPHB2 tyrosine kinase receptor function.

Eph receptor tyrosine kinases participate in a variety of normal and pathogenic processes during development and throughout adulthood. This versatility is likely facilitated by the ability of Eph receptors to signal through diverse cellular signalling pathways: primarily by controlling cytoskeletal dynamics, but also by regulating cellular growth, proliferation, and survival. Despite many proteins linked to these signalling pathways interacting with Eph receptors, the specific mechanisms behind such links and their coordination remain to be elucidated. In a proteomics screen for novel EPHB2 multi-effector proteins, we identified human MYC binding protein 2 (MYCBP2 or PAM or Phr1). MYCBP2 is a large signalling hub involved in diverse processes such as neuronal connectivity, synaptic growth, cell division, neuronal survival, and protein ubiquitination. Our biochemical experiments demonstrate that the formation of a complex containing EPHB2 and MYCBP2 is facilitated by FBXO45, a protein known to select substrates for MYCBP2 ubiquitin ligase activity. Formation of the MYCBP2-EPHB2 complex does not require EPHB2 tyrosine kinase activity and is destabilised by binding of ephrin-B ligands, suggesting that the MYCBP2-EPHB2 association is a prelude to EPHB2 signalling. Paradoxically, the loss of MYCBP2 results in increased ubiquitination of EPHB2 and a decrease of its protein levels suggesting that MYCBP2 stabilises EPHB2. Commensurate with this effect, our cellular experiments reveal that MYCBP2 is essential for efficient EPHB2 signalling responses in cell lines and primary neurons. Finally, our genetic studies in C. elegans provide in vivo evidence that the ephrin receptor VAB-1 displays genetic interactions with known MYCBP2 binding proteins. Together, our results align with the similarity of neurodevelopmental phenotypes caused by MYCBP2 and EPHB2 loss of function, and couple EPHB2 to a signaling effector that controls diverse cellular functions.

Optimized protocol for in vivo affinity purification proteomics and biochemistry using C. elegans.

We present an optimized protocol for in vivo affinity purification proteomics and biochemistry using the model organism C. elegans. We describe steps for target tagging, large-scale culture, affinity purification using a cryomill, mass spectrometry and validation of candidate binding proteins. Our approach has proven successful for identifying protein-protein interactions and signaling networks with verified functional relevance. Our protocol is also suitable for biochemical evaluation of protein-protein interactions in vivo. For complete details on the use and execution of this protocol, please refer to Crawley et al.,1 Giles et al.,2 and Desbois et al.3.

Loss-of-function variants in MYCBP2 cause neurobehavioural phenotypes and corpus callosum defects.

The corpus callosum is a bundle of axon fibres that connects the two hemispheres of the brain. Neurodevelopmental disorders that feature dysgenesis of the corpus callosum as a core phenotype offer a valuable window into pathology derived from abnormal axon development. Here, we describe a cohort of eight patients with a neurodevelopmental disorder characterized by a range of deficits including corpus callosum abnormalities, developmental delay, intellectual disability, epilepsy and autistic features. Each patient harboured a distinct de novo variant in MYCBP2, a gene encoding an atypical really interesting new gene (RING) ubiquitin ligase and signalling hub with evolutionarily conserved functions in axon development. We used CRISPR/Cas9 gene editing to introduce disease-associated variants into conserved residues in the Caenorhabditis elegans MYCBP2 orthologue, RPM-1, and evaluated functional outcomes in vivo. Consistent with variable phenotypes in patients with MYCBP2 variants, C. elegans carrying the corresponding human mutations in rpm-1 displayed axonal and behavioural abnormalities including altered habituation. Furthermore, abnormal axonal accumulation of the autophagy marker LGG-1/LC3 occurred in variants that affect RPM-1 ubiquitin ligase activity. Functional genetic outcomes from anatomical, cell biological and behavioural readouts indicate that MYCBP2 variants are likely to result in loss of function. Collectively, our results from multiple human patients and CRISPR gene editing with an in vivo animal model support a direct link between MYCBP2 and a human neurodevelopmental spectrum disorder that we term, MYCBP2-related developmental delay with corpus callosum defects (MDCD).

Ubiquitin ligase activity inhibits Cdk5 to control axon termination.

The Cdk5 kinase plays prominent roles in nervous system development, plasticity, behavior and disease. It also has important, non-neuronal functions in cancer, the immune system and insulin secretion. At present, we do not fully understand negative regulatory mechanisms that restrict Cdk5. Here, we use Caenorhabditis elegans to show that CDK-5 is inhibited by the RPM-1/FSN-1 ubiquitin ligase complex. This atypical RING ubiquitin ligase is conserved from C. elegans through mammals. Our finding originated from unbiased, in vivo affinity purification proteomics, which identified CDK-5 as a putative RPM-1 substrate. CRISPR-based, native biochemistry showed that CDK-5 interacts with the RPM-1/FSN-1 ubiquitin ligase complex. A CRISPR engineered RPM-1 substrate 'trap' enriched CDK-5 binding, which was mediated by the FSN-1 substrate recognition module. To test the functional genetic relationship between the RPM-1/FSN-1 ubiquitin ligase complex and CDK-5, we evaluated axon termination in mechanosensory neurons and motor neurons. Our results indicate that RPM-1/FSN-1 ubiquitin ligase activity restricts CDK-5 to control axon termination. Collectively, these proteomic, biochemical and genetic results increase our understanding of mechanisms that restrain Cdk5 in the nervous system.

O-GlcNAc transferase OGT-1 and the ubiquitin ligase EEL-1 modulate seizure susceptibility in C. elegans.

Neurodevelopmental disorders such as epilepsy and autism have been linked to an imbalance of excitation and inhibition (E/I) in the central nervous system. The simplicity and tractability of C. elegans allows our electroconvulsive seizure (ES) assay to be used as a behavioral readout of the locomotor circuit and neuronal function. C. elegans possess conserved nervous system features such as gamma-aminobutyric acid (GABA) and GABA receptors in inhibitory neurotransmission, and acetylcholine (Ach) and acetylcholine receptors in excitatory neurotransmission. Our previously published data has shown that decreasing inhibition in the motor circuit, via GABAergic manipulation, will extend the time of locomotor recovery following electroshock. Similarly, mutations in a HECT E3 ubiquitin ligase called EEL-1 leads to impaired GABAergic transmission, E/I imbalance and altered sensitivity to electroshock. Mutations in the human ortholog of EEL-1, called HUWE1, are associated with both syndromic and non-syndromic intellectual disability. Both EEL-1 and its previously established binding protein, OGT-1, are expressed in GABAergic motor neurons, localize to GABAergic presynaptic terminals, and function in parallel to regulate GABA neuron function. In this study, we tested behavioral responses to electroshock in wildtype, ogt-1, eel-1 and ogt-1; eel-1 double mutants. Both ogt-1 and eel-1 null mutants have decreased inhibitory GABAergic neuron function and increased electroshock sensitivity. Consistent with EEL-1 and OGT-1 functioning in parallel pathways, ogt-1; eel-1 double mutants showed enhanced electroshock susceptibility. Expression of OGT-1 in the C. elegans nervous system rescued enhanced electroshock defects in ogt-1; eel-1 double mutants. Application of a GABA agonist, Baclofen, decreased electroshock susceptibility in all animals. Our C. elegans electroconvulsive seizure assay was the first to model a human X-linked Intellectual Disability (XLID) associated with epilepsy and suggests a potential novel role for the OGT-1/EEL-1 complex in seizure susceptibility.

Autophagy is inhibited by ubiquitin ligase activity in the nervous system.

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.

Genetic behavioral screen identifies an orphan anti-opioid system.

Opioids target the µ-opioid receptor (MOR) to produce unrivaled pain management, but their addictive properties can lead to severe abuse. We developed a whole-animal behavioral platform for unbiased discovery of genes influencing opioid responsiveness. Using forward genetics in Caenorhabditis elegans, we identified a conserved orphan receptor, GPR139, with anti-opioid activity. GPR139 is coexpressed with MOR in opioid-sensitive brain circuits, binds to MOR, and inhibits signaling to heterotrimeric guanine nucleotide-binding proteins (G proteins). Deletion of GPR139 in mice enhanced opioid-induced inhibition of neuronal firing to modulate morphine-induced analgesia, reward, and withdrawal. Thus, GPR139 could be a useful target for increasing opioid safety. These results also demonstrate the potential of C. elegans as a scalable platform for genetic discovery of G protein-coupled receptor signaling principles.

A complex containing the O-GlcNAc transferase OGT-1 and the ubiquitin ligase EEL-1 regulates GABA neuron function.

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.

The HECT Family Ubiquitin Ligase EEL-1 Regulates Neuronal Function and Development.

Genetic changes in the HECT ubiquitin ligase HUWE1 are associated with intellectual disability, but it remains unknown whether HUWE1 functions in post-mitotic neurons to affect circuit function. Using genetics, pharmacology, and electrophysiology, we show that EEL-1, the HUWE1 ortholog in C. elegans, preferentially regulates GABAergic presynaptic transmission. Decreasing or increasing EEL-1 function alters GABAergic transmission and the excitatory/inhibitory (E/I) balance in the worm motor circuit, which leads to impaired locomotion and increased sensitivity to electroshock. Furthermore, multiple mutations associated with intellectual disability impair EEL-1 function. Although synaptic transmission defects did not result from abnormal synapse formation, sensitizing genetic backgrounds revealed that EEL-1 functions in the same pathway as the RING family ubiquitin ligase RPM-1 to regulate synapse formation and axon termination. These findings from a simple model circuit provide insight into the molecular mechanisms required to obtain E/I balance and could have implications for the link between HUWE1 and intellectual disability.

Developmental Function of the PHR Protein RPM-1 Is Required for Learning in Caenorhabditis elegans.

The PAM/Highwire/RPM-1 (PHR) proteins are signaling hubs that function as important regulators of neural development. Loss of function in Caenorhabditis elegans rpm-1 and Drosophila Highwire results in failed axon termination, inappropriate axon targeting, and abnormal synapse formation. Despite broad expression in the nervous system and relatively dramatic defects in synapse formation and axon development, very mild abnormalities in behavior have been found in animals lacking PHR protein function. Therefore, we hypothesized that large defects in behavior might only be detected in scenarios in which evoked, prolonged circuit function is required, or in which behavioral plasticity occurs. Using quantitative approaches in C. elegans, we found that rpm-1 loss-of-function mutants have relatively mild abnormalities in exploratory locomotion, but have large defects in evoked responses to harsh touch and learning associated with tap habituation. We explored the nature of the severe habituation defects in rpm-1 mutants further. To address what part of the habituation circuit was impaired in rpm-1 mutants, we performed rescue analysis with promoters for different neurons. Our findings indicate that RPM-1 function in the mechanosensory neurons affects habituation. Transgenic expression of RPM-1 in adult animals failed to rescue habituation defects, consistent with developmental defects in rpm-1 mutants resulting in impaired habituation. Genetic analysis showed that other regulators of neuronal development that function in the rpm-1 pathway (including glo-4, fsn-1, and dlk-1) also affected habituation. Overall, our findings suggest that developmental defects in rpm-1 mutants manifest most prominently in behaviors that require protracted or plastic circuit function, such as learning.

Neuronal development in Caenorhabditis elegans is regulated by inhibition of an MLK MAP kinase pathway.

We show that loss-of-function mutations in kinases of the MLK-1 pathway (mlk-1, mek-1, and kgb-1/jnk) function cell-autonomously in neurons to suppress defects in synapse formation and axon termination caused by rpm-1 loss of function. Our genetic analysis also suggests that the phosphatase PPM-1, like RPM-1, is a potential inhibitor of kinases in the MLK-1 pathway.

Identification of a peptide inhibitor of the RPM-1 · FSN-1 ubiquitin ligase complex.

The Pam/Highwire/RPM-1 (PHR) proteins include: Caenorhabditis elegans RPM-1 (Regulator of Presynaptic Morphology 1), Drosophila Highwire, and murine Phr1. These important regulators of neuronal development function in synapse formation, axon guidance, and axon termination. In mature neurons the PHR proteins also regulate axon degeneration and regeneration. PHR proteins function, in part, through an ubiquitin ligase complex that includes the F-box protein FSN-1 in C. elegans and Fbxo45 in mammals. At present, the structure-function relationships that govern formation of this complex are poorly understood. We cloned 9 individual domains that compose the entire RPM-1 protein sequence and found a single domain centrally located in RPM-1 that is sufficient for binding to FSN-1. Deletion analysis further refined FSN-1 binding to a conserved 97-amino acid region of RPM-1. Mutagenesis identified several conserved motifs and individual amino acids that mediate this interaction. Transgenic overexpression of this recombinant peptide, which we refer to as the RPM-1·FSN-1 complex inhibitory peptide (RIP), yields similar phenotypes and enhancer effects to loss of function in fsn-1. Defects caused by transgenic RIP were suppressed by loss of function in the dlk-1 MAP3K and were alleviated by point mutations that reduce binding to FSN-1. These findings suggest that RIP specifically inhibits the interaction between RPM-1 and FSN-1 in vivo, thereby blocking formation of a functional ubiquitin ligase complex. Our results are consistent with the FSN-1 binding domain of RPM-1 recruiting FSN-1 and a target protein, such as DLK-1, whereas the RING-H2 domain of RPM-1 ubiquitinates the target.

The Nesprin family member ANC-1 regulates synapse formation and axon termination by functioning in a pathway with RPM-1 and ß-Catenin.

Mutations in Nesprin-1 and 2 (also called Syne-1 and 2) are associated with numerous diseases including autism, cerebellar ataxia, cancer, and Emery-Dreifuss muscular dystrophy. Nesprin-1 and 2 have conserved orthologs in flies and worms called MSP-300 and abnormal nuclear Anchorage 1 (ANC-1), respectively. The Nesprin protein family mediates nuclear and organelle anchorage and positioning. In the nervous system, the only known function of Nesprin-1 and 2 is in regulation of neurogenesis and neural migration. It remains unclear if Nesprin-1 and 2 regulate other functions in neurons. Using a proteomic approach in C. elegans, we have found that ANC-1 binds to the Regulator of Presynaptic Morphology 1 (RPM-1). RPM-1 is part of a conserved family of signaling molecules called Pam/Highwire/RPM-1 (PHR) proteins that are important regulators of neuronal development. We have found that ANC-1, like RPM-1, regulates axon termination and synapse formation. Our genetic analysis indicates that ANC-1 functions via the ß-catenin BAR-1, and the ANC-1/BAR-1 pathway functions cell autonomously, downstream of RPM-1 to regulate neuronal development. Further, ANC-1 binding to the nucleus is required for its function in axon termination and synapse formation. We identify variable roles for four different Wnts (LIN-44, EGL-20, CWN-1 and CWN-2) that function through BAR-1 to regulate axon termination. Our study highlights an emerging, broad role for ANC-1 in neuronal development, and unveils a new and unexpected mechanism by which RPM-1 functions.

RPM-1 is localized to distinct subcellular compartments and regulates axon length in GABAergic motor neurons.

BACKGROUND: The PAM/Highwire/RPM-1 (PHR) proteins are conserved signaling proteins that regulate axon length and synapse formation during development. Loss of function in Caenorhabditis elegans rpm-1 results in axon termination and synapse formation defects in the mechanosensory neurons. An explanation for why these two phenotypes are observed in a single neuronal cell has remained absent. Further, it is uncertain whether the axon termination phenotypes observed in the mechanosensory neurons of rpm-1 mutants are unique to this specific type of neuron, or more widespread defects that occur with loss of function in rpm-1. RESULTS: Here, we show that RPM-1 is localized to both the mature axon tip and the presynaptic terminals of individual motor neurons and individual mechanosensory neurons. Genetic analysis indicated that GABAergic motor neurons, like the mechanosensory neurons, have both synapse formation and axon termination defects in rpm-1 mutants. RPM-1 functions in parallel with the active zone component SYD-2 (Liprin) to regulate not only synapse formation, but also axon termination in motor neurons. Our analysis of rpm-1-/-; syd-2-/- double mutants also revealed a role for RPM-1 in axon extension. The MAP3K DLK-1 partly mediated RPM-1 function in both axon termination and axon extension, and the relative role of DLK-1 was dictated by the anatomical location of the neuron in question. CONCLUSIONS: Our findings show that axon termination defects are a core phenotype caused by loss of function in rpm-1, and not unique to the mechanosensory neurons. We show in motor neurons and in mechanosensory neurons that RPM-1 is localized to multiple, distinct subcellular compartments in a single cell. Thus, RPM-1 might be differentially regulated or RPM-1 might differentially control signals in distinct subcellular compartments to regulate multiple developmental outcomes in a single neuron. Our findings provide further support for the previously proposed model that PHR proteins function to coordinate axon outgrowth and termination with synapse formation.

RPM-1 uses both ubiquitin ligase and phosphatase-based mechanisms to regulate DLK-1 during neuronal development.

The Pam/Highwire/RPM-1 (PHR) proteins are key regulators of neuronal development that function in axon extension and guidance, termination of axon outgrowth, and synapse formation. Outside of development, the PHR proteins also regulate axon regeneration and Wallerian degeneration. The PHR proteins function in part by acting as ubiquitin ligases that degrade the Dual Leucine zipper-bearing Kinase (DLK). Here, we show that the Caenorhabditis elegans PHR protein, Regulator of Presynaptic Morphology 1 (RPM-1), also utilizes a phosphatase-based mechanism to regulate DLK-1. Using mass spectrometry, we identified Protein Phosphatase Magnesium/Manganese dependent 2 (PPM-2) as a novel RPM-1 binding protein. Genetic, transgenic, and biochemical studies indicated that PPM-2 functions coordinately with the ubiquitin ligase activity of RPM-1 and the F-box protein FSN-1 to negatively regulate DLK-1. PPM-2 acts on S874 of DLK-1, a residue implicated in regulation of DLK-1 binding to a short, inhibitory isoform of DLK-1 (DLK-1S). Our study demonstrates that PHR proteins function through both phosphatase and ubiquitin ligase mechanisms to inhibit DLK. Thus, PHR proteins are potentially more accurate and sensitive regulators of DLK than originally thought. Our results also highlight an important and expanding role for the PP2C phosphatase family in neuronal development.

The C. elegans developmental timing protein LIN-42 regulates diapause in response to environmental cues.

Environmental conditions can have a major impact on developmental progression in animals. For example, when C. elegans larvae encounter harsh conditions they can reversibly halt the passage of developmental time by forming a long-lived dauer larva at the end of the second larval stage. Here, we show that the period homolog lin-42, known to control developmental time, also acts as a component of a switch that mediates dauer entry. Loss of lin-42 function renders animals hypersensitive to dauer formation under stressful conditions, whereas misexpression of lin-42 in the pre-dauer stage inhibits dauer formation, indicating that lin-42 acts as a negative regulator of this life history decision. These phenotypes place LIN-42 in opposition to the ligand-free form of the nuclear receptor DAF-12, which indirectly senses environmental conditions and helps to integrate external cues into developmental decisions. Mutations that impair DAF-12 ligand binding are exquisitely sensitive to the absence of lin-42, whereas overexpression of LIN-42 can suppress the dauer constitutive phenotype of a ligand-insensitive daf-12 mutant, suggesting that LIN-42 and DAF-12 are intimate partners in controlling the decision to become a dauer larva. The functional outputs of Period family proteins and nuclear receptors also converge in other organisms, suggesting that the relationship between lin-42 and daf-12 represents an ancient genetic framework for responding to environmental stimuli.