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 transgenic fluorescent protein markers. Our past studies have provided insights into synaptogenesis and axon guidance in the developing nervous system. Our recent work has begun to address the regulation of synapses in the mature nervous system and the ability of injured neurons to regenerate and repair damage.
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 and are hallmarks of synapses. By screening for mutants with altered SV distribution, we discovered that the conserved protein SYD-2, a member of the liprin-α 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 is localized to presynaptic membranes and 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. In contrast, a gain-of-function syd-2 mutation 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. Furthermore, our recent studies using biochemistry and three-dimensional electron microscopy tomography show that SYD-2 proteins possess distinct oligomerizing activities to assemble an AZ into a sophisticated nanostructure. These studies provide insights into the molecular network of presynaptic AZ assembly that is conserved throughout evolution.
Regulation of Presynaptic Architecture by the Conserved Giant Ubiquitin Ligase 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 are highly expressed in the nervous systems and have been shown to regulate neuronal development at multiple steps. In mature neurons, RPM-1 is localized to the perisynaptic region, a unique region in the presynaptic terminal. In rpm-1–null mutants, many synapses exhibit an altered architecture: the ratio of synaptic vesicle to presynaptic density is reduced. In addition, some axons overextend beyond normal termination sites.
We have taken a combinatorial approach using genetics and biochemistry to dissect the signaling pathways regulated by RPM-1. 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 among rpm-1(lf) mutants for second-site mutations that eliminate or reduce the activity of candidate 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, the p38-like MAP kinase PMK-3, and the MAPKAP2 MAK-2. Inactivation of each of these kinases suppresses rpm-1(lf) synaptic defects, whereas elevated expression or constitutive activation of the kinases causes synaptic defects resembling rpm-1(lf). The 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. Thus, 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.
Our study further reveals an in vivo function for a previously unknown MAP kinase cascade in presynaptic development. What is the signaling output of the DLK-1/MKK-4/PMK-3/MAK-2 cascade? In our recent studies, we have identified a transcription factor that is a member of the basic–leucine zipper (bZip) protein family as a key target of the MAP kinases. Using live imaging and axotomy, we demonstrated that the mRNA of this bZip protein is present in axons and near synapses, and that the mRNA can be induced to be translated locally in a manner that is dependent on DLK-1. These studies reveal the potential importance of local protein synthesis in synapse formation and maintenance.
Regulation of Locomotion Circuit
Beyond the formation of synapses lie many unanswered questions about how a functional synapse is integrated and regulated within neuronal networks. We have used the C. elegans locomotor circuit to investigate the coordination between the excitatory and inhibitory activities. By characterizing a genetic mutant that harbors a gain-of-function mutation in a neuronal nicotinic acetylcholine receptor, ACR-2, we uncovered a regulation of excitation and inhibition balance in maintaining the sinusoidal movement of the animal. The acr-2(gf) mutant exhibits epileptic-like body contractions. Using genetics and electrophysiology, we find that the ACR-2 receptor acts in the cholinergic motor neurons to maintain their excitability. Intriguingly, hyperexcitation of the cholinergic neurons by acr-2(gf) leads to an anti-homeostatic effect on the downstream inhibitory neurons, resulting in a net overexcitation output from the motor circuit. This acr-2(gf) effect mimics the physiological state of epilepsy. The molecular nature of the acr-2(gf) mutation is nearly identical to several disease-associated mutations in human acetylcholine receptors. Our ongoing studies focus on identification of novel regulators of the network excitation and inhibition imbalance, including ion homeostasis and neuropeptide modulation. These observations will provide mechanistic insight to the search for effective management of disease conditions.
Nerve Regeneration Mechanisms of C. elegans Neurons
The limited ability of mature neurons to regenerate upon injury is an enigma. Such limitations severely impede our ability to repair damaged nerves and to recover function after injury. We have developed a laser-assisted axotomy methodology to sever adult C. elegans neurons, and have observed that the injured axons exhibit neuron-type, neurite-type, and stage-dependent regenerative responses (Movie). The injured proximal axon stumps frequently re-initiate growth and elongate in an error-prone, misguided manner. Using a genetically encoded calcium sensor, we find that the regrowth is correlated with calcium transients that are triggered by axotomy. Moreover, we show that the calcium transients, together with elevating cAMP and protein kinase A signaling, promote early formation of growth cones and subsequent growth rate.
We have performed a large-scale genetic screen to uncover novel and conserved pathways. A key pathway involves the conserved MAPKKK DLK-1, which we identified in studies of synapse formation in development, is essential for regenerative responses in wild type and in mutants with elevated cAMP, whereas increasing dlk-1 enhances growth extent of injured axons. Recent studies from other researchers indicate the function of the DLK pathway in axon regeneration is conserved in Drosophila and mammals. By in-depth analysis of dlk-1 mutant alleles we recently discovered a novel mechanism that controls DLK-1 activation in vivo; the mechanism is likely conserved in mammalian homologs. More recently, we reported a novel role of ribosomal S6 kinase in repressing axon regrowth. Our studies in C. elegans will provide insights to general understanding of the key steps that a mature neuron must overcome for full functional recovery after injury.
Development of a New Optogenetic Method for Manipulation of Molecules and Cells
Optical manipulation of cells and molecules using genetically encoded tags (optogenetics) is revolutionizing neurobiology. In the past three years, we have exploited a new optogenetic reagent, Mini Singlet Oxygen Generator (miniSOG), in a variety of biological contexts. MiniSOG was created by Roger Tsien's lab (HHMI, University of California, San Diego), with the initial purpose of enabling correlative light and electron microscopy. Under blue light excitation, miniSOG produces green fluorescence and generates reactive oxygen species (ROS) that allow visualization of molecules by EM. In collaborative studies with the Tsien lab and the lab of Mark Ellisman (UCSD), we demonstrated that miniSOG could be used for in vivo labeling of organelles and molecules. Taking advantage of the light-inducible ROS-producing ability of miniSOG, we subsequently developed several new applications for inducible ROS-mediated killing of cells and molecules. In combination with other technologies, miniSOG has become a versatile agent to aid functional studies of many cellular processes.
Grants from the National Institutes of Health provided partial support for portions of this work.
As of August 12, 2014