Scientific Background: Mechanisms of neurotransmission:

During synaptic transmission, electrical activity causes a Ca2+ influx into the nerve terminal which triggers exocytosis of synaptic vesicles loaded with transmitter. After fusion with the presynaptic membrane, vesicles reform by an endocytotic recycling mechanism. Postsynaptic membranes have high densities of neurotransmitter receptors tightly localized at the site of the synapse. Neurotransmitter binding to these receptors increases the conductance of the membrane to specific ions and thus, electrical signals are communicated from pre- to postsynaptic cells. Subsequent interaction between synaptic partners determines the extent of innervation, number of synaptic contacts and the efficiency of synaptic transmission. All these essential features of synaptic transmission have been confirmed in a number of vertebrate and invertebrate systems at both NMJ and central synapses. Thus, the mechanisms of neurotransmission appear universally conserved.

To develop an accurate molecular model of neurotransmission we need progress in three areas of research: 1) a complete list of the proteins involved in synaptic function, 2) an understanding of the biochemical interactions and activities of these proteins, and 3) genetic perturbation studies of these proteins to deduce the normal functions of these molecules. In recent years, progress into the molecular mechanisms of synaptic function has been enormous, primarily due to the biochemical purification and classification of proteins in the specialized NMJs of the Torpedo electric organ and synaptic connections in the mammalian brain . These synaptic components include synaptic vesicle (SV) proteins (e.g. Synaptotagmin, Synaptobrevin, Synaptophysin, Synapsins), proteins associated with SVs (e.g. Amphiphysin, Dynamin, CaM kinases), synaptic plasma membrane proteins (e.g. Syntaxins, Neurexins, SNAP-25) and associated cytosolic proteins (e.g. Complexins, SNAPs, NSF). Extensive biochemical analyses of these proteins have generated intricate models of the molecular machinery responsible for excitation-secretion coupling and neurotransmission at the synapse. Many of the identified synaptic proteins have been proposed to fulfill distinctive roles in the excitation-secretion coupling and SV recovery mechanisms underlying synaptic transmission. These classifications are based on the proteinÕs location, biochemical properties and interactions in in vitro assays. The following figure shows a simplified "consensus model" of molecular events during transmission:

The above figure highlights several known proteins and protein-protein interactions occurring during the SV cycle of synaptic transmission. The distribution and availability of vesicles in the SV pool is regulated through protein interactions with the cytoskeleton. For example, the Synapsins are prominent phospho-proteins which are believed to regulate the availability of vesicles clustered near the SV fusion site. SVs are believed to be translocated to the plasma membrane (PM) through a process involving the Rab3 cycle. Prior to docking, the PM protein Syntaxin is complexed to the cytosolic protein Munc18 and a separate complex contains the SV proteins Synaptobrevin and Synaptophysin. At docking, these complexes break apart, and SNAP-25 forms a complex with Syntaxin to form a high-affinity binding site for Synaptobrevin. These three proteins form the synaptic core complex (SCC). The SCC acts as a receptor for SNAPs which, in turn, bind NSF. NSF acts to cross-link the complex and through ATP hydrolysis is believed to prime the two membranes into a "hemifused state". The SV protein Synaptotagmin is thought to then act as a Ca2+ -sensor and complete fusion through an interaction with Syntaxin. After fusion, Synaptotagmin is believed to nucleate clathrin coat assembly by acting as an AP2 complex receptor. Dynamin binds to the AP2 complex to drive endocytosis, the vesicle is recovered and matures to start the process again. This intricate model is based primarily on elegant biochemical and pharmacological work coupled to in vitro assays. The question is: How far is this model correct? Does this model represent the entire mechanism of the SV cycle or are there elements still missing? To test both the validity and completeness of this model, it is essential to experimentally assay its predictions in living synapses by removing individual proteins and assaying the functional consequences of these alterations.

To date, such genetic perturbation studies have lagged behind the biochemical work. The genetic approach has two important aspects: 1) it allows an unbiased screen for all mutations affecting synapse function in order to generate a complete list of essential synaptic proteins, many of which are likely to have been missed with biochemical and pharmacological approaches, and 2) it allows direct assay of a molecule's function in vivo. Ideally, one would like to identify all of the components of the synapse and assay their role by systematically altering or removing each of them in turn. Considerable progress has been made in these analyses by using reverse genetics to remove/alter synaptic proteins identified by biochemical means. This work has been most extensive and successful in the Drosophila system and also using C. elegans (64) and the mouse reverse genetics system. To complement this reverse genetic approach, it is essential that we use forward genetic screens both to uncover the full complement of synaptic components and to systematically mutate each of them. There are few systems in which such a rigorous and comprehensive analysis can be made. However, the use of genetic screens to uncover genes that encode essential elements of complex mechanisms has proved an extremely powerful method, both in C. elegans and in Drosophila. The genetic approach provides an objective assessment of the number of functions required for any process and is an essential step towards a systematic molecular analysis of the mechanisms concerned. In the case of synaptic mechanisms, this approach requires that genetic screens be applied to a synapse which is accessible for analysis of the wild-type and mutant function during both synaptic assembly and operation. Drosophila is the strongest candidate for such an approach since it combines highly developed molecular genetics technology with access to a well-characterized synapse amenable to detailed anatomical and functional assays.

Synaptic communication forms the basis of all information transfer within the nervous system and between the nervous system and the body musculature. The regulation of synaptic connections plays roles in a multitude of processes ranging from neural circuit formation to the control of behavior and coordinated movement to higher brain function such as learning and memory. Synapses are also a prominent site of neuronal dysfunction, such as occurs in inherited neurological disease. Centrally, diseases affecting the synapse include Epilepsy and Parkinson's Disease. Peripherally, at the neuromuscular junction (NMJ), diseases of the synapse include Myasthenia and several types of Muscular Dystrophy. Finally, the regulation of synaptic mechanisms is a central aspect of neuronal regeneration following injury or stroke, for example brain trauma or the loss of a limb. Thus, understanding the basis of synapse formation and function is of primary importance in our investigation of fundamental neuronal mechanisms, in our search for cures for many prominent forms of neurological disease and in the treatment of neuronal damage caused by injury.

We have initiated a systematic investigation of the cellular, molecular and genetic mechanisms of synapse function. This approach requires two conditions; 1) a genetic system is which the genome can be systematically screened for mutations in synaptic genes, and 2) a system containing an accessible synapse which can be monitored with sophisticated functional and anatomical assays. The only system which meets these requirements is the Drosophila NMJ. Drosophila is a classic genetic system which has repeatedly led the way into the genetic dissection of conserved biological mechanisms. Moreover, we have shown that the NMJ develops and functions using conserved synaptic mechanisms and that single synaptic gene mutations can be assayed in fine detail using both electrophysiological and ultrastructural assays. In this research project, we propose to combine forward and reverse genetic approaches to the synapse. Forward screens will give an unbiased survey of all the genes required for synapse formation and function. Reverse genetics allows one to target synaptic genes already implicated at the synapse from earlier biochemical or pharmacological work. A combination of these approaches will lead to a global understanding of the genetic and molecular mechanisms of synapse function.

Our research has two specific aims. First, to saturate the Drosophila genome for mutations perturbing synaptic function. We are currently engaged in a saturation mutagenesis for essential synaptic genes on the third chromosome of Drosophila, representing approximately 40% of the genome. Isolated mutants are being placed in phenotypic and complementation groups and each group assayed for synaptic dysfunction using functional and anatomical assays. Genes of interested will be cloned and molecularly characterized. Second, the analysis of synaptic mutants isolated by reverse genetics. To date, we have seven genes (Synaptotagmin, Synaptobrevin, Rop (n-sec1/Munc-18 homologue), Shibire (Dynamin homologue), Leonardo (14-3-3 protein homologue), Syntaxin, Cysteine String Protein) known to play prominent roles in neurotransmission. Several more are currently being characterized. Our objective is to define the mutant phenotypes of such genes in order to elucidate the molecular machinery of excitation-secretion coupling at the synapse. My aim is to use this powerful genetic approach to span the distance between molecules, synaptic function and higher behavior.

I have developed the first system for the systematic genetic analysis of synapse formation and genes essential for synapse function. A major gap in our investigation of the synapse was the inability to analyze genetic mutations that effect synapse formation or are required for synaptic function. Such an approach requires experimental access to a synapse as it develops in vivo in a well-defined genetic organism. I have developed the Drosophila NMJ to fill this essential experimental niche. I developed all the tools and knowledge required to make this system useful. A list of these advances include the following:

1. I developed techniques for the acute dissection of the Drosophila embryo at any stage of synaptic development. These techniques allow experimental access to the developing synapse in vivo.

2. I developed a culture system for the extended culture of the Drosophila embryo. These techniques allow synaptogenesis to be monitored in an accessible in vitro system.

3. I developed all the electrophysiological recording techniques in the Drosophila embryo, including a wide range of techniques required to stimulate and record from the developing synapse in vivo.

4. I developed ultrastructural techniques for the developing Drosophila synapse. These techniques include both immuno-scanning electron microscope (SEM) and transmission electron microscope (TEM) technology.

Using these techniques I initiated the first systematic investigation of synapse formation and function using a genetic approach. My advances include the following:

1. I established a complete timetable of normal synaptogenesis in Drosophila.

2. I used genetic mutants to define cellular interactions during synaptogenesis. These included mutants to selectively remove the pre- or postsynaptic cell and mutants to increase or decrease the amount of electrical activity during development.

3. I was the first to analyze the phenotype of an essential synaptic gene in any system (synaptotagmin, 1994). I have since characterized six essential synaptic gene mutants, more than have been analyzed in any other system.

In summary, I have founded my own field for the genetic dissection of synapse formation and function, I now intend to use the tools and knowledge I have developed to conduct the first systematic genetic investigation of the synapse.

Our research focus is on the cellular, molecular and genetic mechanisms of synapse function. It is our intention to systematically examine the mechanisms of the synapse using a combination of saturation mutageneses to uncover novel genes and reverse genetic approaches aimed at known synaptic genes. We propose to uncover synaptic genes in the classic genetic system of Drosophila and study their function through detailed functional and anatomical assays of synaptic dysfunction in genetic mutants. The synaptic system we will use to pursue this genetic approach is the Drosophila neuromuscular junction (NMJ), an accessible synapse with well-studied developmental and functional properties (see above section) [1-3]. We will work primarily on the embryonic NMJ, since all genes essential for synapse function necessarily mutate to result in early lethality [4,5]. We have previously shown that the embryonic synapse in amenable to functional analyses and that single synaptic gene mutations cause definitive defects in this novel preparation (see above section). We now propose to apply the sophisticated genetic techniques of Drosophila to the comprehensive dissection of synaptic mechanisms. In the long-term, we intend to systematically mutate the Drosophila genome to uncover and analyze the complete genetic and molecular pathways involved in the function of the synapse.
This research project has two specific aims, each of which contribute to the thorough genetic investigation of synaptic mechanisms. Aim #1 is designed to systematically uncover unknown synaptic genes. Aim #2 is designed to characterize the in vivo function of known synaptic genes:

1. Saturation Mutagenesis of the Third Chromosome of Drosophila Our immediate aim is to saturate the third chromosome of Drosophila for mutations in genes essential to synapse function. This chromosome comprises approximately 40% of the Drosophila genome. Mutations will be isolated in a series of screens and then analyzed at the NMJ using functional and anatomical assays. Genes of interest will be mapped on the chromosome and molecularly characterized. The intention of this aim is to compile a complete list of the essential synaptic genes on the third chromosome and systematically describe their molecular nature and the role of each in the synapse. In the future, we plan to extend this approach to the entire Drosophila genome.

2. Reverse Genetic Approach: Characterization of Mutants in Known Genes To complement the saturation mutagenesis aimed at uncovering novel synaptic genes, we will continue to expand on my analysis and characterization of known synaptic genes. We have a large and growing catalogue of synaptic proteins implicated in neurotransmission which have been isolated by biochemical or pharmacological methods [6,7]. Many of the protein homologues have been identified in Drosophila and the genes mutated to assay in vivo function [5]. The remainder of the known synaptic genes are actively being targeted for mutational analysis in Drosophila. The detailed analyses of these mutants should allow us to describe the molecular machinery underlying neurotransmission function. Priorities in the research include the functional and anatomical characterization of a number of newly-isolated presynaptic function mutants and the construction and analysis of multiply-mutant animals.

Specific Aim #1: A. Rationale The formation and function of synapses has been highly conserved between different types of synapses (e.g. cholinergic vs. glutamatergic) and between species during evolution (e.g. Drosophila vs. human) [4,5,8-11]. Therefore, information gained from any particular system is likely to be directly applicable to all synapses. We are conducting a saturation mutagenesis of the Drosophila 3rd chromosome to uncover these conserved synaptic genes. The mutagenesis is being conducted with a non-selective chemical mutagen (EMS) and will continue until every mutable locus has sustained two or more independent hits (estimated at 7-10,000 mutant lines). The screen will be conducted in a parent line marked with muscle and nerve reporter gene constructs to allow rapid screening and elimination of mutants with morphological abnormalities. Isolation of mutants will involve a 5-nested screening protocol: 1) a screen for pre-adult lethality (using adult visible markers), 2) a screen for embryonic/early postembryonic lethality (hatching assays), 3) a screen for gross morphology (muscle/neural reporter gene constructs, antibody stains), 4) a screen for movement/coordinated locomotion and 5) functional screens using physiological techniques. Selected lines will be embryonic/early postembryonic lethals with normal morphology but movement/locomotion defects attributable to synaptic dysfunction.

B. Experimental Plan Genetic mutants will be generated and bred using established Drosophila techniques. The selection will be for dysfunctional neuromuscular mutants using the nested 5-protocol screening procedure established in our preliminary studies. Mutants of interest will then be characterized using a combination of functional and anatomical assays. In earlier work, I have developed whole-cell patch clamp techniques for recording from embryonic lethal mutant synapses [12-14]. I have also developed a range of techniques to stimulate and assay synapse functional characteristics [15,16]. To complement these functional studies, I have developed a range of LM, SEM and TEM techniques to assay the morphology and ultrastructure of the synapse [12,16,17]. A combination of these techniques will allow me to isolate novel synaptic mutants and pinpoint the pre- or post-synaptic location of the genetic defect. Novel synaptic mutants will be complementation tested and mapped to a chromosomal location on the giant polytene salivary chromosomes using standard Drosophila methods. Novel mutations with interesting functional defects will be cloned and molecularly characterized using well established techniques.

Specific Aim #2: A. Rationale Synapse function has been highly conserved through evolution [9,18,19]. Individual synaptic genes show 70+% identity between Drosophila and higher vertebrates and their functional roles appear highly similar [5,20]. We propose to continue with the reverse genetic approach of isolating Drosophila homologues of important synaptic genes and then mutating them to examine their functional role in synaptic transmission. We will focus my energies on the phenotypic description of mutant defects using functional and anatomical studies at the embryonic NMJ [4,5]. This work will involve both the analysis of newly generated mutants and the analysis of previously identified mutants (particularly in multiply-mutant combinations). Mutations in the synaptic genes encoding Neurexin and a Rab3-interacting gene have recently been identified. A priority in our research will be the characterization of null mutations in these two genes at the embryonic NMJ.

B. Preliminary Work I have previously characterized a large number of genes required for neuromuscular synapse function [1,4,5].This work has defined a pool of mutants to use as tools for the further dissection of synaptic mechanisms. It will be particular important to look at multiply mutant combinations of these genes, an approach which has not yet been done in this or any other system. In regards to the two specified new synaptic genes (Neurexin and a Rab3A-interacting gene), the initial reverse molecular genetic work has already been completed or is near completion. Both genes mutate to cause severe movement impairment and early lethality. These results, combined with previous work in other systems, indicate that both genes are prime candidates for essential components of the neurotransmission machinery.

C. Experimental Plan This research effort is being divided into two components: 1) characterization of newly defined synaptic mutants, and 2) the detailed analysis of previously isolated synapse function mutants. For the first effort, new mutants will be analyzed as they are isolated. As outlined above, we are currently involved in the characterization of a number of new synapse function mutants. All mutants will be characterized using the embryonic NMJ preparation with a combination of functional and anatomical assays. For the second effort, it will be particularly important to generate multiply-mutant animals to uncover the nature of molecular interactions at the synapse. For example, animals mutant for both Synaptotagmin and Synaptobrevin, Synaptotagmin and Cysteine String Protein, Synaptotagmin and Shibire (Dynamin), Syntaxin and Synaptobrevin, and a number of other mutant combinations. These lines will need to be assayed using both functional and anatomical tests as was initial done for each single mutation. In addition, many of these previously isolated mutants still require much more detailed analyses. In particular, EM studies and imaging of ion flux dynamics.

The aim of this research program is to increase substantially the number of known genes involved in the construction and working of a synapse. Such genes can be isolated, identified and their functional roles rapidly determined using the Drosophila system which I have developed. A great deal of evidence has shown that the molecular and genetic mechanisms of the synapse have been highly conserved through evolution [4,5,8,11,21-13]. Therefore, the information gained from Drosophila will be directly applicable to man. This research is of primary importance to several areas of biomedical research. First, for the treatment of inherited neuromuscular diseases affecting the NMJ such as Muscular Dystrophy and Myasthenia. Second, for the treatment of central synaptic disorders such as Epilepsy and Parkinson's Disease. Third, for the treatment of neuronal damage following paralysis, stroke or injury. Finally, for the understanding of the synaptic mechanisms of learning and memory and their disorder resulting from injury, disease or aging.

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