Project Summary

Throughout the nervous system there is evidence that the refinement and modulation of neural circuitry is driven not only by synapse formation, but also by the regulated disassembly of previously functional synaptic connections (Katz and Shatz, 1996; Sanes and Lichtman, 1999; Eaton and Davis, 2003). Very little is known about the molecular mechanisms that regulate and achieve synapse disassembly in the nervous system of any organism.  We have developed high-throughput assays for synapse disassembly that, combined with our genome-scale dsRNA collection, allows us to screen the genome for genes that, when knocked down or disrupted, cause synapse disassembly. Using this strategy as well as classical forward genetic approaches we hope to define a core cellular program that controls synapse stability versus disassembly.  As discussed below, the genes we identify may have relevance to the cause and progression of neuromuscular degenerative disease in humans. 

An Assay for Synapse Disassembly at the Drosophila NMJ.  We have previously published an assay for synapse disassembly at the Drosophila NMJ (Eaton et al., 2002).  The basis for this assay is diagrammed below (Figure 1).  Briefly, the postsynaptic membrane folds at the Drosophila NMJ (termed SSR) are composed of highly compact muscle membrane folds up to several microns thick (Lahey et al., 1994).  The gradual assembly of the SSR and proteins that localize to this structure require the presence of the presynaptic nerve terminal (Guan et al., 1996; Thomas et al., 2000).  Therefore, the SSR and proteins that localize to this structure will be present only at sites where the nerve terminal resides, or where it has recently resided.  Presynaptic retraction is more rapid than the disassembly of the SSR.  Thus, sites where SSR markers are present without opposing presynaptic markers represent sites of presynaptic nerve terminal retraction (Eaton et al., 2002). This assay has now been tested using presynaptic membrane markers, presynaptic cytoskeletal markers, synaptic vesicle markers, endocytic protein markers, and presynaptic transmembrane proteins. We have also confirmed synapse retractions using electron microscopy and synaptic electrophysiology.

Synaptic Disassembly in Drosophila and the Mechanisms of Human Neuromuscular Neurodegenerative Disease.  One of the first genes that we identified in our genome-scale RNAi screen was the Arp-1 subunit of the dynactin complex.  The dynactin complex along with dynein mediates most of the retrograde axonal transport in neurons.  Disruption of dynactin function has recently been implicated in the cause and etiology of amyotrophic lateral sclerosis (ALS).  ALS is a debilitating, late onset neurodegenerative disease in humans. Approximately 20% of familial cases of ALS are caused by disruption of the superoxide dismutase gene, SOD-1.  The remaining familial and all sporadic cases (more than 80% of all ALS cases) are of unknown cause.  A role for impaired dynein/dynactin-mediated retrograde transport in ALS has recently been suggested by disruption of dynactin in mouse motoneurons (LaMonte et al., 2002).  These mice share the phenotypic hallmarks of human ALS that are also observed in the SOD-1 knockout mouse. Interestingly, there is also a parallel between the SOD-1 mouse and disruption of dynactin at the Drosophila NMJ.  In both systems, synapse destabilization and retraction are observed (Eaton et al., 2002; J. Lichtman, Soc. Neurosci Abstract, 2002).  Finally, it was recently found that a lower motor disease in humans showed linkage to a region of chromosome 2p13 that contains a mutation in the dynactin complex (Puls et al., 2003). These data suggest the important possibility that the genes that we identify in Drosophila may have implications for the isolation of motor disease-related genes in humans.

RESEARCH GOALS.

1. Identification of Candidate Disease Related Genes.  Using the “synaptic footprint” as a quantitative, high throughput assay we are using forward genetics and genome-scale RNAi to identify new genes that when knocked down or eliminated cause inappropriate synapse disassembly.  This analysis is in early stages and has led to the identification of at least three new genes implicated in this process including trophic signaling molecules and synaptic adaptor proteins.

2.  Identification of Mutations that Prevent Synapse Disassembly.  Our second goal is to use forward genetic screens to identify mutations in genes that can suppress synapse disassembly.  We are taking existing mutations implicated in human ALS (for example) and are hoping to identify genes that, when mutated or overexpressed, prevent synapse retraction and elimination.  To date we have identified four different genetic conditions that can suppress severe synapse disassembly. These preliminary data demonstrate that it is possible to suppress synapse disassembly in this system.  These “suppressors” may represent interesting candidates for future studies in mammalian system and may represent candidates for future intervention in neuromuscular degenerative disease.

3.  Investigating Known Human Disease Genes.  Several human genes have been linked to neuromuscular degenerative diseases.  However, the mechanisms by which the loss of these genes lead to neurodegeneration is generally unknown.  Many of these genes have homologues in Drosophila.  We plan to perform genetic epistasis tests to determine whether genes isolated in our genetic screens can be linked to the biology of these human disease genes.