J. Troy Littleton
Synaptic Plasticity in Drosophila
The focus of our laboratoryDs work is on the elucidation of the molecular mechanisms underlying synapse formation, function and plasticity. We are combining molecular biology, protein biochemistry, electrophysiology, electron microscopy and imaging approaches with Drosophila genetics to investigate molecular mechanisms involved in neuronal signaling. Current genetic approaches in the lab include the identification and characterization of novel temperature-sensitive paralytic mutants in Drosophila as a tool to identify and study new components of neuronal signaling pathways. In addition, we are engaged in reverse genetic approaches for structure-function studies of known synaptic proteins, including the Drosophila synaptotagmin and syntaxin families. A separate area of study in the lab involves the use of Drosophila as a transgenic model for the study of trinucleotide repeat diseases and the causes and consequences of epilepsy.
Characterization of Drosophila Temperature-Sensitive Paralytic Mutations:
The primary form of intercellular communication within the nervous system is mediated by chemical transmission at synapses. Upon propagation of an action potential into the nerve terminal, there is an influx of calcium through voltage-activated calcium channels that triggers the fusion of docked synaptic vesicles with the pre-synaptic membrane. Neurotransmitters are then released into the synaptic cleft and subsequently bind to post-synaptic receptors. Synaptic transmission has evolved as a highly specialized form of cellular vesicle trafficking capable of rapid calcium triggered exocytotic cycles. We have conducted behavioral screens to identify mutants in synaptic transmission in Drosophila by screening for EMS induced mutations that cause temperature sensitive paralysis at elevated temperatures. The goal of the screen is to generate thermoliable proteins that when brought to the non-permissive temperature will block an essential function required for synaptic transmission. Many novel temper-ature-sensitive paralytic mutations have now been generated and are currently being analyzed. We have previously characterized two paralytic mutations, comatose and syntaxin. The neuronal SNARE complex is formed via the interaction of the vesicular SNARE synaptobrevin with the target membrane SNAREs syntaxin and SNAP-25. Disassembly of the SNARE complex by NSF has been proposed to drive lipid bilayer fusion, an essential feature of eukaryotic membrane trafficking. We have isolated a Drosophila temperature- sensitive paralytic mutation in syntaxin that rapidly blocks synaptic transmission at non-permissive temperatures. This paralytic mutation results from a single amino acid change in the H3 domain of syntaxin that specifically and selectively decreases binding to synaptobrevin and abolishes assembly of the 7S SNARE complex. Temperature-sensitive paralytic mutations in NSF (comatose) also block synaptic transmission, but over a much slower time course. NSF function is exhausted in an activity-dependent fashion and results in a large increase in undissociated SNARE complexes. Both mutations result in an increase in the number of morphologically docked vesicles. Thus, assembly and disassembly of the SNARE complex is required for synaptic transmission in vivo, with a syntaxin-synaptobrevin interaction requirement for vesicle fusion and a role for NSF in maintaining disassembled SNARE complexes during vesicular trafficking. We are continuing this line of research to identify enhancers and suppressors of the syntaxin mutant in order to identify new proteins involved in vesicle fusion. Similar analysis of additional temperature-sensitive paralytic mutations should yield novel insights into mechanisms of membrane excitability and synapse formation and function.
Characterization of the Role of the Synaptotagmin Family in Synapse Function and Seizure Modulation:
Among the modifications that make vesicle trafficking at the synapse different from other forms of cellular membrane trafficking is the ability to rapidly trigger fusion in response to calcium influx, the requirement to hold vesicles in a fusion ready state until a calcium signal, and to rapidly endocytose the vesicular membrane and vesicle proteins for reuse in additional rounds of fusion. We are currently investigating the function of synaptotagmin, which may be involved in all three of these neuronal specific requirements for synaptic vesicle trafficking. Synaptotagmin is a synaptic vesicle integral membrane protein that contains two calcium binding repeats within the cytosolic portion of the protein. We have now generated over 20 mutations within synaptotagmin and are initiating structure-function studies of synaptotagmin I. We have also identified eight new synaptotagmins in Drosophila, including a homolog of synaptotagmin IV, an intermediate-early gene upregu-lated in mammalian seizure models. Recent studies have shown that chronic depolarization or seizure activity in mammals results in the up-regulation of a distinct and unusual isoform of the synaptotagmin family, synaptotagmin IV. Drosophila synaptotagmin IV is enriched on synaptic vesicles and contains an evolutionary conserved aspartate to serine substitution that abolishes its ability to bind membranes in response to calcium influx. Synaptotagmin IV forms hetero-oligomers with synaptotagmin I that result in synaptotagmin-clusters that cannot effectively penetrate lipid bilayers and that are less efficient at coupling calcium to secretion in vivo: up-regulation of synaptotagmin IV, but not synap-totagmin I, decreases evoked neurotransmission. These findings indicate that modulation of expression of synaptotagmins with different calcium-binding affinities can lead to heteromultimers with novel properties that regulate the efficiency of excitation-secretion coupling in vivo and represent a novel molecular mechanism for synaptic plasticity. Thus, the upregulation of synapto-tagmin IV in seizure models may have evolved as an adaptive mechanism to decrease neuronal excitability. Further studies of synaptotagmin IV as well as other genes upregulated in Drosophila seizure models is likely to identify new modulatory synaptic mechanisms that control synaptic output during seizure activity and that are also likely to be used as a mechanism to modulate synaptic plasticity during learning and memory.
Generation and Characterization of a Drosophila Model of Trinucleotide Repeat Disease:
Trinucleotide repeat diseases include a variety of human disorders including HuntingtinDs disease. We are currently investigating how expansion of a CAG repeat and the resulting polyglutamine stretch causes neuronal dysfunction. We have generated transgenic Drosophila overexpressing human huntingtin proteins with CAG repeat lengths from 0 to 120. These lines will be used to investigate the effect of huntingtin overexpression on synapse function, apoptosis, and neuronal development. In addition, we are generating huntingtin constructs to overexpress the protein in tissue-specific patterns. This work will allow us to begin suppressor screens for proteins that might be able to block the ability of abnormal huntingtin proteins to cause cell death. These approaches should generate insights into pathways that result in neuronal dysfunction caused by polyglutamine repeats in neurodegenerative disorders.