A brain is composed of cells connected to one another at synapses. Our goal is to build a molecular model for the synapse—to understand how the proteins work together to form a nanomachine. We hope that this model will lead to insights into ways that the output of the synapse can be modulated.
To understand how the synapse functions, we are dissecting synaptic function in the simplest organism with a well-defined nervous system—the nematode Caenorhabditis elegans. Our strategy is to first identify worms with defects in synaptic proteins. We do this either by forward genetic screens for worm mutants with behavioral defects or by generating directed mutations in synaptic genes identified by others. We then use biochemistry, electrophysiology, and fluorescence and electron microscopy to examine the role of the protein in the synapse from as many angles as we can. Next we build a model for the function of the protein, based on inferences from the mutant phenotype. Predictions for structural variants are made from these models and tested in transgenic animals. These results can be used as a springboard for studies in other systems, linking biochemical models of protein function and studies of the vertebrate brain.
Our current research can be classified into three broad categories: neurotransmitters, exocytosis, and endocytosis.
My lab was founded on exploring the inhibitory neurotransmitter GABA, starting with the identification of the vesicular GABA transporter. Because the mutant phenotype of this gene was so distinctive we screened for other genes that could be mutated to this phenotype. In addition to inhibitory receptors, we identified two excitatory GABA receptors. These receptors demonstrate previously unappreciated evolutionary plasticity in one of the most well studied of the neurotransmitter systems. From similar screens we identified mutations in a plasma membrane H+ ion transporter on one cell and a proton-gated ion channel, evolved from an acetylcholine receptor, on a second cell. When calcium levels rise in the first cell, it releases protons, which are sensed by the second cell, a muscle, which depolarizes and contracts. Together these two molecules form a two-molecule, cell-cell signaling complex for the smallest neurotransmitter—a proton.
We are continuing to identify other novel molecules that can act as neurotransmitters in C. elegans. For example, we have recently shown that betaine, a molecule similar to acetylcholine, is the physiological ligand for a neurotransmitter receptor that is of interest because it is the target of a new class of nematode-killing drugs.
Synaptic Vesicle Exocytosis
Neurotransmitters are released when synaptic vesicles dock to the plasma membrane and then fuse. We have characterized the role of SNAREs, UNC-13, and complexin in synaptic vesicle docking. At the synapse, membrane fusion is driven by the formation of a SNARE complex composed of syntaxin and SNAP-25 on the plasma membrane and synaptobrevin on the vesicle. We generated animals that have GABA neurons completely devoid of syntaxin. Mutant neurons lacked synaptic transmission and lacked docked vesicles, showing that syntaxin is essential for both vesicle docking and fusion. UNC-13 is a protein that binds syntaxin in all nervous systems. We have shown that UNC-13 promotes the open conformation of syntaxin, either directly or indirectly, and thereby allows synaptic vesicles to dock at the membrane. In related experiments, we have demonstrated that the UNC-13-like protein CAPS activates syntaxin during dense-core vesicle docking.
Complexin is a small protein that binds to the SNARE complex. The role of complexin at the synapse is particularly controversial: mouse knockouts suggest that complexin is required for fusion; fly knockouts suggest that complexin blocks fusion. In C. elegans, complexin is bifunctional: it both inhibits and stimulates fusion. We propose that the binding of complexin to the SNARE complex stabilizes docked vesicles in a partially wound state, blocking both fusion and undocking.
Synaptic Vesicle Endocytosis
Synaptic vesicle proteins and membrane must be recovered after fusing to the synaptic membrane. From our studies of endocytosis, there are four significant conclusions. First, a protein involved in exocytosis—the calcium sensor synaptotagmin—is involved in endocytosis. Second, novel adaptors recruit synaptic vesicle proteins to endocytic structures. Third, the classic protein adaptor AP2 is not required to recruit specific synaptic vesicle proteins but is involved in membrane recruitment. Fourth, clathrin, the workhorse of endocytosis, is not required for synaptic vesicle endocytosis. These surprising results suggest that synaptotagmin and adaptor proteins may make major contributions to membrane endocytosis. Clathrin, for which the process of clathrin-mediated endocytosis was named, may play little or no role in synaptic vesicle endocytosis.
Recently, we have developed a technique for freezing and fixing both mouse and worm neurons in the milliseconds after synaptic vesicle release. We then image the neurons with an electron microscope to see how the membranes are moving during endocytosis. We have discovered that in both organisms there is a rapid endocytosis of membrane from the surface, a process we call ultrafast endocytosis. Currently we are using genetic and pharmacological techniques to determine the molecular players in this process.
Grants from the National Institutes of Health and National Science Foundation provided partial support for these projects.
As of February 7, 2014