Overview
The computational power of the brain depends on synaptic connections that link together billions of neurons. The focus of my laboratory's work is to understand the mechanisms by which neurons form synaptic connections, how synapses transmit information, and how synapses change during learning and memory. To complement this basic research in neuroscience, we also study how alterations in neuronal signaling underlie several neurological diseases, including epilepsy and Huntington’s Disease. We combine molecular biology, protein biochemistry, microarray technology, electrophysiology, and imaging approaches with Drosophila genetics to address these questions. Moving beyond genomic data to ultimately understand how proteins specify the distinctive signaling properties of neurons and enable them to interconnect into computational circuits that dictate behavior are major goals for the next decade. Despite the dramatic differences in complexity between Drosophila and humans, genomic analysis has confirmed that key neuronal proteins and the functional mechanisms they govern are remarkably similar. As such, we are attempting to elucidate the mechanisms underlying synapse formation, function and plasticity using Drosophila as a model system. By characterizing how neurons integrate synaptic signals and modulate synaptic growth and strength, we hope to bridge the gap between molecular components of the synapse and the physiological responses they mediate. To accomplish these goals, the lab has developed a research plan that encompasses a genetic and functional analysis of several aspects of synaptic biology as indicated below:

 

Research Summary
1. Genetic Dissection of Synapse Formation and Synaptic Growth.
Axonal sprouting and synaptic rewiring are key regulators of neuronal plasticity in the developing and adult brain. Similar to many species, modulation of synapse formation in Drosophila has been implicated in learning and memory. To elucidate the mechanisms underlying synapse formation and subsequent synaptic growth, as well as to characterize how the regulation of these events modulates plasticity and behavior, we use forward and reverse genetic approaches to identify molecular pathways essential to the process. As a starting point, we have characterized the role of neuronal activity in synapse formation using the Drosophila glutamatergic neuromuscular junction (NMJ) as a model. Disruptions of postsynaptic glutamate receptors or postsynaptic vesicle trafficking cause defects in presynaptic nerve terminal differentiation, suggesting calcium influx through glutamate receptors induces presynaptic growth via the release of retrograde signals from postsynaptic vesicles.

Consistent with this model, overexpression of proteins that enhance postsynaptic retrograde signaling results in increased sprouting of synaptic boutons. We are currently characterizing the molecular components that regulate postsynaptic vesicle fusion, as well as the retrograde signals involved. We hypothesize that calcium influx into postsynaptic cells induces secretion of retrograde signals from postsynaptic vesicles, activating a cAMP pathway in presynaptic terminals that modulates synaptic growth in a Hebbian input/output-specific manner.

To identify additional neuronal pathways that control synapse formation and synaptic growth, we have conducted forward genetic screens for mutants that disrupt the process. In addition to retrograde signaling, we have found that presynaptic calcium influx through N-type channels participates in synaptic growth via signaling pathways that are distinct from those that mediate neurotransmitter release. The opening of presynaptic N-type channels during robust synaptic activity may allow calcium to modulate sprouting mechanisms that locally control synaptic remodeling. Several cell adhesion proteins and synaptic growth regulators reside in peri-active regions adjacent to the active zone where presynaptic calcium channels localize.

We have recently identified one such peri-active zone protein, termed nervous wreck (nwk), that modulates synaptic growth. NWK interacts with WASP, and together the complex regulates synaptic growth and activity by controlling actin polymerization in presynaptic terminals. Further genetic analysis of synaptic growth mutants is underway to identify molecular pathways that modulate synaptic connectivity and alter behavioral output.

2.Genetic Dissection of Neurotransmitter Release.
To complement our studies on synapse formation, we have also examined how synaptic connections function following assembly. Katz and colleagues established the hypothesis that calcium influx into the presynaptic nerve terminal triggers neurotransmitter release. Current models for synaptic vesicle exocytosis propose that calcium triggers fusion through activation of the SNARE complex. Although SNAREs are essential for synaptic transmission, fusion via reconstituted SNARE proteins occurs over slow timescales and is calcium-independent, contrasting with synaptic transmission, where SNARE assembly and subsequent fusion is rapid and calcium-triggered.

The search for synaptic calcium sensors that regulate SNARE-dependent fusion has largely focused on the synaptotagmins, transmembrane proteins containing tandem calcium binding C2 domains (C2A and C2B). To characterize the mechanisms that couple calcium influx to rapid and synchronous synaptic vesicle fusion we are analyzing Drosophila synaptotagmin I mutants deficient in specific interactions mediated by its two calcium-binding C2 domains. In the absence of synaptotagmin I, synchronous release is abolished and a kinetically distinct delayed asynchronous release pathway is uncovered. Mutant synapses containing only the C2A domain of synaptotagmin partially recover synchronous fusion, but have an abolished calcium cooperativity. Mutants that disrupt calcium sensing by the C2B domain have synchronous release with normal calcium cooperativity, but with reduced release probability.

Our results indicate synaptotagmin is the major calcium sensor for evoked release and functions By using targeted genetic approaches and forward genetic screens for neurotransmitter release mutants, we expect to gain new insights into the molecular pathways that mediate synaptic communication between neurons.

3. Modeling Neurological Disease in Drosophila. With the realization that many basic neuronal mechanisms are conserved between Drosophila and humans, we have used Drosophila to model several neurological diseases. Huntington’s disease is an autosomal dominant neurodegenerative disorder caused by expression of a polyglutamine tract in the huntingtin protein that forms intracellular aggregates. To date, pathways leading from polyglutamine expansion to disease pathogenesis remain unknown. We have generated transgenic Drosophila expressing the mutant human huntingtin gene and have reproduced many aspects of the neurological damage that occurs in humans.

While expression of normal human huntingtin has no discernible effect on behavior, lifespan or neuronal morphology in Drosophila, pan-neuronal expression of Htt-Q128 leads to progressive loss of motor coordination, decreased lifespan and time-dependent formation of huntingtin aggregates specifically in the cytoplasm and neurites. Huntingtin aggregates sequester other expanded polyglutamine proteins in the cytoplasm and lead to synaptic aggregate accumulation and disruption of axonal transport. In contrast, Drosophila expressing an expanded polyglutamine tract alone, or an expanded polyglutamine tract in the context of the spinocerebellar ataxia type 3 protein, display only nuclear aggregates and do not disrupt axonal trafficking.

Our findings indicate that non-nuclear events induced by cytoplasmic huntingtin aggregation may play a central role in the progressive neurodegeneration observed in Huntington’s disease. We are now characterizing how huntingtin-mediated disruption of axonal transport causes toxicity and how it can be prevented. We hope that finding ways to cure our Drosophila model of Huntington’s Disease will reveal conserved mechanisms that might be eventual targets for therapy in humans.

Additional strategies are being used to generate and characterize Drosophila models of epilepsy and muscular dystrophy. In particular, we have focused extensively on generating Drosophila epilepsy models. To identify neuronal dysfunctions that cause epilepsy, we have conducted behavioral screens for temperature-sensitive (TS) paralytic mutations induced by EMS that result in seizures. To date, 79 seizure-inducing mutations that define 27 complementation groups have been identified in large-scale screens of homozygous viable lines generated in the lab.

We find that increasing neuronal activity drives overproliferation of synaptic connections, indicating activity-dependent rewiring occurs in Drosophila as observed in mammalian epilepsy models. To analyze genome-wide alterations in gene expression resulting from the transcriptional program activated by enhanced neuronal activity, we performed DNA microarray analysis using 7 Drosophila seizure models and four seizure induction paradigms, allowing us to compare over 100 different activity states in the fly brain.

Approximately 200 genes were found to display substantial alterations in expression following changes in neuronal excitability. These 200 genes encode proteins putatively involved in neuronal connectivity, membrane excitability, and neuronal signaling. Several of these activity-regulated proteins are predicted to function in synapse formation and have been analyzed by RNA interference (RNAi) to characterize their role in modulating synaptic connectivity. These 200 loci represent exciting candidate genes that may regulate synaptic morphology and synaptic transmission in an activity-dependent manner.

Together with our basic neuroscience research, these studies will expand our understanding of the mechanisms of synapse formation, function and plasticity. They will also provide important insights into how the nervous system functions at the cellular level, allowing us to integrate this information into the framework of ultimately understanding how neuronal ensembles mediate behavior, and how neurological diseases disrupt these processes.

 

Selected Publications
Montana, E.S. & Littleton, J.T. (2006) Expression profiling of a hypercontraction-induced myopathy in Drosophila suggests a compensatory cytoskeletal remodeling response. J. Biol. Chem. 281, 8100-8109.

Yoshihara, M., Adolfsen, B., Galle, K & Littleton J.T. (2005) Retrograde signaling by Syt 4 induces presynaptic release and synapse-specific growth. Science 310, 858-863.

Guan, Z., Saraswati, S., Adolfsen, B. & Littleton, J.T. (2005) Genome-wide transcriptional changes associated with enhanced activity in the Drosophila nervous system. Neuron 48, 91-107.

Adolfsen, B., Saraswati, S., Yoshihara, M. & Littleton, J.T (2004) Synaptotagmins are trafficked to distinct subcellular domains including the postsynaptic compartment. J. Cell Biology 166, 249-260.

Wang, P., Saraswati, S., Guan, Z., Watkins, C., Wurtman, R.J. & Littleton, J.T. (2004) A Drosophila temperature-sensitive seizure mutant in phosphoglycerate kinase disrupts ATP generation and alters synaptic function. J. Neuroscience 24, 4518-4529.

Coyle, I., Koh, Y-H., Lee, W.C.M, Slind, J., Fergestad, T., Littleton, J.T. & Ganetzky, B. (2004) Nervous Wreck, an SH3 adapter protein that interacts with Wsp, regulates synaptic growth in Drosophila. Neuron 41, 521-534.

Lee, W.C., Yoshihara, M. & Littleton, J.T. (2004) Cytoplasmic aggregates trap polyglutamine-containing proteins and block axonal transport in a Drosophila model of Huntington’s Disease. PNAS 101, 3224-3229.

Montana, E.S. & Littleton, J.T. (2004) Characterization of a hypercontraction induced myopathy in Drosophila caused by mutations in Mhc. J. Cell Biology 164, 1045-1054.

Search PubMed for Littleton Lab publications.

 

 

 

Synaptotagmin labeling (white) at neuromuscular junctions of Drosophila melanogaster.