ABSTRACT
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.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , GABAergic Neurons/metabolism , Ubiquitin-Protein Ligases/metabolism , Aldicarb/toxicity , Animals , Animals, Genetically Modified/metabolism , Axons/metabolism , Caenorhabditis elegans Proteins/antagonists & inhibitors , Caenorhabditis elegans Proteins/genetics , Electroshock , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , Hypersensitivity/etiology , Locomotion/drug effects , Mutagenesis, Site-Directed , Presynaptic Terminals/metabolism , RNA Interference , Signal Transduction , Synapses/metabolism , Synaptic Transmission/drug effects , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/geneticsABSTRACT
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.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/genetics , JNK Mitogen-Activated Protein Kinases/genetics , MAP Kinase Kinase 1/genetics , MAP Kinase Signaling System , Neurogenesis , Animals , Axons/metabolism , Caenorhabditis elegans/growth & development , Caenorhabditis elegans Proteins/metabolism , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Kinase 1/metabolism , Synapses/metabolismABSTRACT
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.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Guanine Nucleotide Exchange Factors/genetics , Microfilament Proteins/genetics , Neurogenesis/genetics , beta Catenin/genetics , Animals , Animals, Genetically Modified , Axons , Caenorhabditis elegans/genetics , Caenorhabditis elegans/growth & development , Caenorhabditis elegans Proteins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Microfilament Proteins/metabolism , Mutation , Neurons/cytology , Neurons/metabolism , beta Catenin/metabolismABSTRACT
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.
Subject(s)
Caenorhabditis elegans Proteins/physiology , Guanine Nucleotide Exchange Factors/physiology , MAP Kinase Kinase Kinases/physiology , Neurogenesis , Phosphoric Monoester Hydrolases/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Axons , Caenorhabditis elegans Proteins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , MAP Kinase Kinase Kinases/metabolism , Myristic Acid/metabolism , Protein BindingABSTRACT
Previous studies in Caenorhabditis elegans showed that RPM-1 (Regulator of Presynaptic Morphology-1) regulates axon termination and synapse formation. To understand the mechanism of how rpm-1 functions, we have used mass spectrometry to identify RPM-1 binding proteins, and have identified RAE-1 (RNA Export protein-1) as an evolutionarily conserved binding partner. We define a RAE-1 binding region in RPM-1, and show that this binding interaction is conserved and also occurs between Rae1 and the human ortholog of RPM-1 called Pam (protein associated with Myc). rae-1 loss of function causes similar axon and synapse defects, and synergizes genetically with two other RPM-1 binding proteins, GLO-4 and FSN-1. Further, we show that RAE-1 colocalizes with RPM-1 in neurons, and that rae-1 functions downstream of rpm-1. These studies establish a novel postmitotic function for rae-1 in neuronal development.
Subject(s)
Axons/physiology , Mechanoreceptors/cytology , Nuclear Matrix-Associated Proteins/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , Synapses/physiology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Motifs/genetics , Amino Acid Sequence/genetics , Animals , Animals, Genetically Modified , Caenorhabditis elegans , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , F-Box Proteins/genetics , F-Box Proteins/metabolism , Gene Expression Regulation/genetics , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , Humans , Immunoprecipitation , Luminescent Proteins/genetics , Mass Spectrometry , Microscopy, Confocal , Molecular Sequence Data , Mutation/genetics , Nuclear Matrix-Associated Proteins/deficiency , Nuclear Matrix-Associated Proteins/genetics , Nucleocytoplasmic Transport Proteins/deficiency , Nucleocytoplasmic Transport Proteins/genetics , Protein Binding/genetics , Signal Transduction/genetics , Synapses/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolismABSTRACT
The PHR (Pam/Highwire/RPM-1) proteins are evolutionarily conserved ubiquitin ligases that regulate axon guidance and synapse formation in Caenorhabditis elegans, Drosophila, zebrafish, and mice. In C. elegans, RPM-1 (Regulator of Presynaptic Morphology-1) functions in synapse formation, axon guidance, axon termination, and postsynaptic GLR-1 trafficking. Acting as an E3 ubiquitin ligase, RPM-1 negatively regulates a MAP kinase pathway that includes: dlk-1, mkk-4, and the p38 MAPK, pmk-3. Here we provide evidence that ppm-1, a serine/threonine phosphatase homologous to human PP2Cα(PPM1A) and PP2Cß(PPM1B) acts as a second negative regulatory mechanism to control the dlk-1 pathway. We show that ppm-1 functions through its phosphatase activity in a parallel genetic pathway with glo-4 and fsn-1 to regulate both synapse formation in the GABAergic motorneurons and axon termination in the mechanosensory neurons. Our transgenic analysis shows that ppm-1 acts downstream of rpm-1 to negatively regulate the DLK-1 pathway, with PPM-1 most likely acting at the level of pmk-3. Our study provides insight into the negative regulatory mechanisms that control the dlk-1 pathway in neurons and demonstrates a new role for the PP2C/PPM phosphatases as regulators of neuronal development.