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The Role of Complexin in Neurotransmitter Release

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The Role of Complexin in Neurotransmitter Release THE ROLE OF COMPLEXIN IN NEUROTRANSMITTER RELEASE Neurons communicate using specialized con- Student Researcher: Joel Fernandez nections called synapses by releasing synaptic Mentor: Jeremy Dittman, Weill Cornell Medical College, New York, NY vesicles (SVs) containing neurotransmitters. How this process is regulated is not completely understood, although some of the key mole- cules have been identified. Complexins are a INTRODUCTION proteins in vivo. Furthermore, many family of protein found primarily in the synaptic mutants have already been nervous system of all animals. We investigated Chemical synapses play a critical identified (a complexin null mutant the role of complexin I (cpx-1) in the nematode role in the function of an animal's among them). C. elegans. We found that cpx-1 null mutants Using this organism, we tested paralyzed far quicker in aldicarb than wild- nervous system. They are the means type animals. Furthermore, the null mutants through which neurons transfer infor- whether complexin plays an inhibitory displayed severe locomotory defects; the null mation. The presynaptic terminal is or facilitating role in neurotransmitter mutants showed about a 70% decrease in pivotal in this exchange. It is responsible release. Furthermore, we wanted to body bends per 20 seconds. Introducing a for the regulated release of vesicles know if a null mutant can be rescued complexin GFP fusion protein into GABA by reintroducing complexin into the motor neurons alone did not rescue the containing neurotransmitters, the chem- mutants; these animals displayed the exact icals which relay messages across synap- animal. defects as the null mutants, suggesting that null ses. How this vital and rapid process is defects are independent of the GABA motor regulated is still a mystery, although neurons. However, putting the same com- many key players have been identified. METHODS plexin backinto all neurons did rescue the null mutants; rescue animals paralyzed on aldicarb The basic fusion machinery is the with a wild-type time course and showed SNARE complex, a complex made of To test the function of complexin in about a 150% increase in body bends than the three proteins: SNAP-25, synaptobre- synaptic exocytosis, we utilized an null mutant. Our data suggest not only that vin, and syntaxin. The complex alone is available C. elegans complexin null complexin plays a critical role in neurotrans- 1 mutant. The mutant allele contains a mitter release but also that complexin is an sufficient to drive membrane fusion. inhibitor of this process. Furthermore, our data However, it is insufficient to drive fast, large deletion including much of the also imply that including a C terminal GFP synchronous release; other proteins are coding region. This mutation is reces- does not impair complexin function. Through needed for this process. sive, as heterozygous animals appear further analysis of complexin, we hope to One of these proteins is complexin. wild-type. better understand the protein's function in regulating synaptic transmission. Complexins are a family of protein In order to test synaptic activity in found primarily in the nervous system the mutant, we used the aldicarb assay. of all animals. Mammals contain four Aldicarb blocks the enzyme acetyl- complexin proteins (CplxI, CplxII, cholinesterase, which is responsible for CplxIII, CplxIV). Complexin is known the breakdown of acetylcholine (ACh) to bind to the SNARE complex.2 in the synaptic cleft. Continued release However,itsfunctionisunknown. of ACh causes prolonged excitation of There is evidence to suggest that com- the muscle, leading to paralysis. Thus, plexin acts as a ``fusion clamp'' and thus increases in ACh levels at the synapse inhibits exocytosis.1 However, recent increase sensitivity to aldicarb. experiments in mice suggest that com- Aldicarb Assays. Approximately 20 plexin in fact facilitates synaptic exocy- wild-type, cpx-1 null mutant, and tosis.3 transgenic rescue animals (per trial) In order to test complexin in vivo, were coded (in order to perform the we used the nematode Caenorhabditis assay blind) and transferred onto 1 mM elegans as knocking out complexin does aldicarb plates. Animals were scored for not kill this model organism. C. elegans paralysis every 10 minutes for two has a simple nervous system made up of hours, beginning 20 minutes after only 302 neurons. Furthermore, C. transferring the animals onto aldicarb elegans has only two complexin genes plates. (cpx-1, cpx-2), of which cpx-1 is an Locomotion Assays. Ten animals ortholog of mammalian CplxI and per genotype were placed on agar plates CplxII. The worm's transparency is containing no food and left at 23u C for helpful in imaging fluorescently-tagged an hour. Body bends were counted per Ethnicity & Disease, Volume 19, Summer 2009 S3-13 Fernandez and Dittman 20 seconds to quantify locomotion compared to the null mutants). This the seemingly contradictory findings de- rates. Five animals were scored from finding supported our aldicarb results. scribed above. There is evidence to believe each plate and three plates were score The aldicarb sensitivity of cpx-1 null that complexin stabilizes the SNARE per genotype. A body bend was counted mutants can also be explained by complex in a primed state to allow for if a point posterior to the pharyngeal decreased GABA release at the neuro- Ca2+-evoked exocytosis instead of asyn- bulb moved right or left relative to the muscular junction (NMJ). If complexin chronous release. Additionally, studies animal's midline.4 Sample size was plays a facilitating role in the GABAer- have shown that the protein synaptotag- recorded in plate averages. gic NMJs removal of complexin de- min 1, which has been described as the creases GABA release, accounting for neuron's calcium sensor, competes with the increased sensitivity to aldicarb and complexin to bind to the SNARE com- locomotion defects. To test whether this plex.5 In this model, calcium-bound RESULTS was the case, complexin was restored in synaptotagmin displaces complexin and GABAergic neurons and assayed for allows for vesicle fusion. Therefore, any We found that the complexin null rescue of aldicarb sensitivity and loco- defects in Ca2+-evoked exocytosis is not mutant paralyzed substantially quicker motion. We found that there was no due to a facilitory role for complexin, but than wild-type animals, suggesting that significant difference in aldicarb sensi- rather a supportive role. complexin inhibits vesicle fusion. tivity or locomotion rates among the In the future, we will employ quan- To ensure that hypersensitivity to GABA-specific rescue animals and the titative in vivo imaging of synaptic aldicarb was indeed due to the loss of cpx-1 null mutants, therefore supporting complexin-GFP to better understand its complexin rather than some other the hypothesis that complexin acts in recruitment and retention during synap- defects, we introduced a full length cholinergic neurons to inhibit transmit- tic activity. Furthermore, we will image rescuing complexin cDNA into null ter release. complexin in various synaptic mutants to mutant animals using a pan-neuronal determine how complexin interacts with promoter. The transgenic animals were other proteins. We will also delete various restored to near wild-type aldicarb DISCUSSION domains of the protein to test the sensitivity, suggesting not only that the function of each domain in behavioral aldicarb phenotype of null mutants is Our results suggest two major con- and imaging assays. complexin dependent, but also that clusions. First, complexin plays an inhib- expressing complexin in neurons alone itory role at synapses in C. elegans. REFERENCES is sufficient to rescue the animals. Second, cpx-1 null mutants can be 1. Melia TJ, Jr. Putting the clamps on membrane We also conducted a behavioral rescued using a cpx-1 GFP fusion protein. fusion: How complexin sets the stage for assay testing rates of locomotion of the calcium-mediated exocytosis. FEBSLett . Our first conclusion is consistent 2007;581:2131±2139. nematode. Locomotion assays provide with previous experiments in mice. 2. Chen X, et al. Three-dimensional structure of another means of assessing the role of Overexpressing complexin in mice hip- the complexin/SNARE complex. Neuron. complexin in the worm nervous system. pocampal cells strongly inhibited exo- 2002;33:397±409. Loss of a critical protein in synaptic cytosis, thus supporting the ``fusion 3. Xue M, et al. Complexins facilitate neurotrans- mitter release at excitatory and inhibitory exocytosis would result in severe loco- 1,5 clamp'' hypothesis. However, recent synapses in mammalian central nervous system. motion defect. We assayed the locomo- experiments describe reduced calcium Proc Natl Acad Sci USA. 2008;105:7875±7880. tion of cpx-1 null mutants to determine (Ca2+)-triggered exocytosis in com- 4. McEwan JM, Madison JM, Dybbs M, Kaplan the severity of nervous system impair- plexin-knockout mice hippocampal JM. Antagonistic regulation of synaptic vesicle ment. Indeed, cpx-1 null mutants dis- cells, suggesting that complexin facili- priming by Tomosyn and UNC-13. Neuron. 2006;51:303±315. played a 70% decrease in locomotion tates exocytosis.3 5. Tang J, et al. A complexin/synaptotagmin 1 (per 20 s), while the transgenic rescue Another possible role for complexin switch controls fast synaptic exocytosis. Cell. animal displayed a 150% increase (as has been described, which could explain 2006;126:1175±1187. S3-14 Ethnicity & Disease, Volume 19, Summer 2009.
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