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Neural Development in C. elegans
Summary: Yishi Jin is interested in understanding the molecular mechanisms controlling wiring and rewiring of the nervous system in the nematode Caenorhabditis elegans.
Signal transduction from neurons to their targets depends on specialized subcellular structures at synaptic junctions. A typical chemical synapse is composed of three compartments: a presynaptic terminal for rapid neurotransmitter release, a postsynaptic site that is rich in neurotransmitter receptors to receive the signal, and a synaptic cleft that bridges the pre- and postsynaptic partners and maintains them in precise registration. The size and organization of synapses vary widely between neuronal types and are modulated by experience. The general processes of synaptogenesis and how such processes give rise to the diversity of synapses in the nervous system are topics under intense investigation.
We use the simple nervous system of the nematode C. elegans to examine how synapses are formed. This nervous system is composed of only 302 neurons, and its entire connectivity is known at the ultrastructural level. Moreover, most of the synaptic connections can be observed in living animals using GFP (green fluorescent protein) markers. Our studies have provided insights into synaptogenesis and axon guidance, and we have recently begun to explore the possibility of studying nerve regeneration in C. elegans.
Regulation of Presynaptic Architecture by the Conserved Giant Molecule RPM-1
The C. elegans RPM-1 (regulator of presynaptic morphology) is a member of a conserved protein family that includes Drosophila Highwire and vertebrate Phr1, Pam, and Esrom. These large proteins contain multiple conserved motifs, including an RLD guanine nucleotide exchange factor (GEF) domain and a RING E3 ubiquitin ligase domain. They have all been implicated in nervous system development. RPM-1 is localized to the perisynaptic region, a unique region in the presynaptic terminal. In rpm-1–null mutants, synapses exhibit an altered architecture: the ratio of synaptic vesicle to presynaptic density is reduced.
We hypothesize that one reason the synapses are improperly formed in rpm-1 mutants is because RPM-1 substrates are not degraded in a timely manner. To identify such substrates, we screened rpm-1(lf) mutants for second-site mutations that eliminate or reduce the activity of the of RPM-1 substrates, therefore restoring synaptic defects of rpm-1(lf) to normal organization. This analysis led us to the identification of a novel MAP kinase cascade composed of the dual leucine zipper–bearing MAPKKK DLK-1, the MAPKK MKK-4, and the p38-like MAP kinase PMK-3. Inactivation of these three kinases suppresses rpm-1(lf) synaptic defects, whereas elevated expression or constitutive activation of the kinases causes synaptic defects resembling rpm-1(lf). The function of this kinase cascade is required presynaptically. In particular, DLK-1/MAPKKK is localized to the synaptic region, and its synaptic abundance is elevated in rpm-1 mutants. Biochemically, RPM-1 can bind DLK-1 and can stimulate ubiquitination of DLK-1. Our study thus reveals an in vivo function for a previously unknown MAP kinase cascade in presynaptic development. The activity of this MAP kinase cascade is negatively controlled by RPM-1 through the regulation of protein stability. This underscores the importance of ubiquitin-mediated regulation in synapse development.
RPM-1 is a giant protein and likely functions through multiple signal transduction pathways. To identify the other pathways interacting with RPM-1, we used a proteomic approach to search for proteins associated with RPM-1. One of the candidates is GLO-4, an RLD-domain–containing GEF protein. We find that GLO-4 colocalizes with RPM-1 at the presynaptic terminals. Loss of function in glo-4 causes synaptic defects partially resembling those in rpm-1 mutants. Furthermore, we show that GLO-4 GEF acts through a Rab GTPase and the AP3 adapter proteins. The GLO/AP3 pathway is positively regulated by RPM-1 to promote vesicular trafficking through late endo-lysosomes. Thus, through a combination of genetic, biochemical, and molecular approaches, we discovered two parallel signaling pathways that function downstream of RPM-1 in synapse formation. Growing evidence supports the hypothesis that these signaling mechanisms are conserved in other organisms and in other cellular processes. (A grant from the National Institutes of Health provided partial support for this project.)
Assembly of Presynaptic Active Zones
The active zones (AZs) at the presynaptic terminals are located at the center of the synaptic vesicle (SV) exocytic zone, promote SV docking, and provide the precise registry between pre- and postsynaptic specializations. AZs display morphologically distinct ultrastructures. By screening for mutants with altered SV distribution, we discovered that the conserved protein SYD-2, a member of the α-liprin protein family, is a key regulator of AZ assembly. Liprins are composed of highly conserved coiled-coil and SAM domains. They were originally identified as binding partners for the LAR receptor protein-tyrosine phosphatase (RPTP). We find that C. elegans LAR, known as PTP-3, is localized to presynaptic membranes. PTP-3 acts with the extracellular matrix protein nidogen to pattern presynaptic terminals. In syd-2(lf) mutants, AZs exhibit a fragmented appearance, causing diffused localization of SVs. Recently, we uncovered an unusual gain-of-function syd-2 mutation that is able to promote synapse assembly in the absence of several upstream regulators. Using this mutation, we have dissected the complex interactions among AZ proteins. This study provides insight into the molecular network of presynaptic AZ assembly. (Grants from National Institutes of Health and the Muscular Dystrophy Association provided partial support for this work.)
Nerve Regeneration Mechanisms of C. elegans Neurons
C. elegans ventral cord motor neurons have been excellent models to study the mechanisms guiding axons to their targets during development of the nervous system. Our early work identified several genes functioning in motor axon repulsion. We have shown that the novel intracellular protein MAX-1 acts in the UNC-5 netrin receptor pathway for long-range repulsion and that the ADAM protein UNC-71 functions with integrins and with UNC-6/netrin to provide local cues at specific choice points.
In our current research, we have examined what kind of cues the regenerating axons in the mature nervous system use. C. elegans axons can be severed using a femtosecond near-infrared laser microsurgery technique. We have found that C. elegans neurons exhibit diverse regenerative responses, depending on the types of neurons, developmental stages, and axotomy sites. Importantly, we have found that ephrin signaling influences the precision of regenerating nerves. (This work was supported in part by grants from the National Institutes of Health.)
GABAergic Neuron Specification
GABA is the major inhibitory neurotransmitter in the nervous system, and GABAergic neurons are essential for brain function. My lab is interested in specification of GABAergic neurons. C. elegans has 26 GABAergic neurons of five classes. Our early work showed that UNC-30 homeodomain protein specifies the type D neurons. Four RME neurons innervate head muscles to control foraging behavior, and are classified as one group. We showed that the C. elegans homolog of the aryl hydrocarbon receptor (AHR) specifies the fate of two RME neurons. Mutations in ahr-1 transform RMEL/R into RMED/V, revealing unexpected differences within this neuronal class. AHR proteins are mainly studied for their roles in toxin response. Our work, together with studies in Drosophila, indicates an ancestral role for these proteins in neuronal fate specification. To define transcriptional profiles of these GABAergic neurons, we used fluorescence-activated cell sorting (FACS) to identify a set of ~250 genes enriched in these neurons. We are combining computation predictions and experimental validation to identify genes essential for GABA neuron diversification. (Grants from the National Institutes of Health and the Packard Foundation provided partial support for this work.)
Last updated: September 18, 2007
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