Neurotoxicity Pathways in Drosophila Models of the Polyglutamine Disorders.

Although polyglutamine expansion diseases are the most common genetically inherited neurodegenerative disorders, the key pathogenic mechanisms that lead to neuronal cell death are unclear. The expansion of a polyglutamine tract in specific proteins is the defining molecular insult, leading to cell-type and region-specific neuronal death. Intraneuronal aggregates of the affected protein can be found in the nucleus and/or cytoplasm and are a hallmark of these disorders. Whether and how aggregation leads to pathology, however, is under debate. In this chapter, we will review some of the key observations using Drosophila models of polyglutamine disorders that have highlighted a host of potential contributing pathologies, including defects in transcription, autophagy, and mitochondrial biology. We will also examine how genetic screening approaches have been used in Drosophila to provide insights into potential therapeutic approaches for polyglutamine disorders.

SNARE-complex disassembly by NSF follows synaptic-vesicle fusion.

Soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE)-mediated fusion of synaptic vesicles with the presynaptic-plasma membrane is essential for communication between neurons. Disassembly of the SNARE complex requires the ATPase N-ethylmaleimide-sensitive fusion protein (NSF). To determine where in the synaptic-vesicle cycle NSF functions, we have undertaken a genetic analysis of comatose (dNSF-1) in Drosophila. Characterization of 16 comatose mutations demonstrates that NSF mediates disassembly of SNARE complexes after synaptic-vesicle fusion. Hypomorphic mutations in NSF cause temperature-sensitive paralysis, whereas null mutations result in lethality. Genetic-interaction studies with para demonstrate that blocking evoked fusion delays the accumulation of assembled SNARE complexes and behavioral paralysis that normally occurs in comatose mutants, indicating NSF activity is not required in the absence of vesicle fusion. In addition, the entire vesicle pool can be depleted in shibire comatose double mutants, demonstrating that NSF activity is not required for the fusion step itself. Multiple rounds of vesicle fusion in the absence of NSF activity poisons neurotransmission by trapping SNAREs into cis-complexes. These data indicate that NSF normally dissociates and recycles SNARE proteins during the interval between exocytosis and endocytosis. In the absence of NSF activity, there are sufficient fusion-competent SNAREs to exocytose both the readily released and the reserve pool of synaptic vesicles.

Genetic and molecular analysis of the synaptotagmin family.

Secretion is a fundamental cellular process used by all eukaryotes to insert proteins into the plasma membrane and transport signaling molecules and intravesicular proteins into the extracellular space. Secretion requires the fusion of two phospholipid bilayers within the cell, an energetically unfavorable process. A conserved repertoire of vesicle-trafficking proteins has evolved that function to overcome this energy barrier and temporally and spatially control membrane fusion within the cell. Within neurons, opening of synaptic calcium channels and subsequent calcium entry triggers synchronous synaptic vesicle exocytosis and neurotransmitter release into the synaptic cleft. After fusion, synaptic vesicles undergo endocytosis, are refilled with neurotransmitter, and return to the vesicle pool for further rounds of cycling. It is within this local synaptic trafficking pathway that the synaptotagmin family of calcium-binding synaptic vesicle proteins has been postulated to function. Here we review the current literature on the function of the synaptotagmin family and discuss the implications for synaptic transmission and membrane trafficking.

synaptotagmin mutants reveal essential functions for the C2B domain in Ca2+-triggered fusion and recycling of synaptic vesicles in vivo.

Synaptotagmin has been proposed to function as a Ca(2+) sensor that regulates synaptic vesicle exocytosis, whereas the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is thought to form the core of a conserved membrane fusion machine. Little is known concerning the functional relationships between synaptotagmin and SNAREs. Here we report that synaptotagmin can facilitate SNARE complex formation in vitro and that synaptotagmin mutations disrupt SNARE complex formation in vivo. Synaptotagmin oligomers efficiently bind SNARE complexes, whereas Ca(2+) acting via synaptotagmin triggers cross-linking of SNARE complexes into dimers. Mutations in Drosophila that delete the C2B domain of synaptotagmin disrupt clathrin AP-2 binding and endocytosis. In contrast, a mutation that blocks Ca(2+)-triggered conformational changes in C2B and diminishes Ca(2+)-triggered synaptotagmin oligomerization results in a postdocking defect in neurotransmitter release and a decrease in SNARE assembly in vivo. These data suggest that Ca(2+)-driven oligomerization via the C2B domain of synaptotagmin may trigger synaptic vesicle fusion via the assembly and clustering of SNARE complexes.

Neurobiology and the Drosophila genome.

The sequencing and annotation of the euchromatin of the Drosophila melanogaster genome provides an important foundation that allows neurobiologists to work back from the complete gene set of neuronal proteins to an eventual understanding of how they function to produce cognition and behavior. Here we provide a brief survey of some of the key insights that have emerged from analyzing the complete gene set in Drosophila. Not surprisingly, both the Caenorhabditis elegans and Drosophila genomes contain a conserved repertoire of neuronal signaling proteins that are also present in mammals. This includes a large number of neuronal cell adhesion receptors, synapse-organizing proteins, ion channels and neurotransmitter receptors, and synaptic vesicle-trafficking proteins. In addition, there are a significant number of fly homologs of human neurological disease loci, suggesting that Drosophila is likely to be an important disease model for human neuropathology in the near future. The experimental analysis of the Drosophila neuronal gene set will provide important insights into how the nervous system functions at the cellular level, allowing the field to integrate this information into the framework of ultimately understanding how neuronal ensembles mediate cognition and behavior.

The C2B domain of synaptotagmin is a Ca(2+)-sensing module essential for exocytosis.

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca(2+) sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a large cytoplasmic domain that contains two C2 domains, C2A and C2B. Multiple Ca(2+) ions bind to the membrane proximal C2A domain. However, it is not known whether the C2B domain also functions as a Ca(2+)-sensing module. Here, we report that Ca(2+) drives conformational changes in the C2B domain of synaptotagmin and triggers the homo- and hetero-oligomerization of multiple isoforms of the protein. These effects of Ca(2)+ are mediated by a set of conserved acidic Ca(2)+ ligands within C2B; neutralization of these residues results in constitutive clustering activity. We addressed the function of oligomerization using a dominant negative approach. Two distinct reagents that block synaptotagmin clustering potently inhibited secretion from semi-intact PC12 cells. Together, these data indicate that the Ca(2)+-driven clustering of the C2B domain of synaptotagmin is an essential step in excitation-secretion coupling. We propose that clustering may regulate the opening or dilation of the exocytotic fusion pore.

Comparative genomics of the eukaryotes.

A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae-and the proteins they are predicted to encode-was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.

Synaptic function modulated by changes in the ratio of synaptotagmin I and IV.

Communication within the nervous system is mediated by Ca2+-triggered fusion of synaptic vesicles with the presynaptic plasma membrane. Genetic and biochemical evidence indicates that synaptotagmin I may function as a Ca2+ sensor in neuronal exocytosis because it can bind Ca2+ and penetrate into lipid bilayers. Chronic depolarization or seizure activity results in the upregulation of a distinct and unusual isoform of the synaptotagmin family, synaptotagmin IV. We have identified a Drosophila homologue of synaptotagmin IV that is enriched on synaptic vesicles and contains an evolutionarily conserved substitution of aspartate to serine that abolishes its ability to bind membranes in response to Ca2+ influx. Synaptotagmin IV forms hetero-oligomers with synaptotagmin I, resulting in synaptotagmin clusters that cannot effectively penetrate lipid bilayers and are less efficient at coupling Ca2+ to secretion in vivo: upregulation of synaptotagmin IV, but not synaptotagmin I, decreases evoked neurotransmission. These findings indicate that modulating the expression of synaptotagmins with different Ca2+-binding affinities can lead to heteromultimers that can regulate the efficiency of excitation-secretion coupling in vivo and represent a new molecular mechanism for synaptic plasticity.

Temperature-sensitive paralytic mutations demonstrate that synaptic exocytosis requires SNARE complex assembly and disassembly.

The neuronal SNARE complex is formed via the interaction of synaptobrevin with syntaxin and SNAP-25. Purified SNARE proteins assemble spontaneously, while disassembly requires the ATPase NSF. Cycles of assembly and disassembly have been proposed to drive lipid bilayer fusion. However, this hypothesis remains to be tested in vivo. We have isolated a Drosophila temperature-sensitive paralytic mutation in syntaxin that rapidly blocks synaptic transmission at nonpermissive temperatures. This paralytic mutation 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 and with the accumulation of syntaxin and SNARE complexes on synaptic vesicles. These results provide in vivo evidence that cycles of assembly and disassembly of SNARE complexes drive membrane trafficking at synapses.

ROP, the Drosophila Sec1 homolog, interacts with syntaxin and regulates neurotransmitter release in a dosage-dependent manner.

The Sec1 family of proteins is thought to function in both non-neuronal and neuronal secretion, although the precise role of this protein family has not been defined. Here, we study the function of ROP, the Drosophila Sec1 homolog, in neurotransmitter release. Electrophysiological analyses of transgenic lines overexpressing ROP and syntaxin, a presynaptic membrane protein, indicate that ROP interacts with syntaxin in vivo. Characterization of four point mutations in ROP shows that they fall into two phenotypic classes. Two mutations cause a dramatic reduction in both evoked and spontaneous neurotransmitter release. In contrast, the other two mutations reveal an increase in evoked neurotransmission. Our data further show that neurotransmission is highly sensitive to the levels of ROP function. Studies on heterozygote animals indicate that half the amount of wild-type ROP results in a dramatic decrease in evoked and spontaneous exocytosis. Taken together, these results suggest that ROP interacts with syntaxin in vivo and is a rate-limiting regulator of exocytosis that performs both positive and inhibitory functions in neurotransmission.

A Drosophila neurexin is required for septate junction and blood-nerve barrier formation and function.

Septate and tight junctions are thought to seal neighboring cells together and to function as barriers between epithelial cells. We have characterized a novel member of the neurexin family, Neurexin IV (NRX), which is localized to septate junctions (SJs) of epithelial and glial cells. NRX is a transmembrane protein with a cytoplasmic domain homologous to glycophorin C, a protein required for anchoring protein 4.1 in the red blood cell. Absence of NRX results in mislocalization of Coracle, a Drosophila protein 4.1 homolog, at SJs and causes dorsal closure defects similar to those observed in coracle mutants. nrx mutant embryos are paralyzed, and electrophysiological studies indicate that the lack of NRX in glial-glial SJs causes a breakdown of the blood-brain barrier. Electron microscopy demonstrates that nrx mutants lack the ladder-like intercellular septa characteristic of pleated SJs (pSJs). These studies identify NRX as the first transmembrane protein of SJ and demonstrate a requirement for NRX in the formation of septate-junction septa and intercellular barriers.

Immunocytochemical analysis of axonal outgrowth in synaptotagmin mutations.

Synaptotagmin is a synaptic vesicle specific protein that binds calcium and phospholipids in vitro and is required for calcium-regulated fusion of synaptic vesicles with the presynaptic membrane. We have examined the possible requirement for synaptotagmin in axonal outgrowth by following neuronal development in Drosophila embryos deficient for the synaptotagmin gene. We find that synaptotagmin is expressed abundantly in axons and growth cones before synapse formation in wild-type embryos. Using antibodies to the intravesicular domain of synaptotagmin to label live embryos, we demonstrate that vesicle populations containing synaptotagmin actively undergo exocytosis during axonogenesis. We have used immunocytochemical techniques to examine the distribution of the axonal protein Fasciclin II, the presynaptic membrane protein syntaxin, and the synaptic vesicle protein cysteine string protein, in synaptotagmin null mutations. The distribution of these proteins is similar in wild-type and synaptotagmin mutant embryos, suggesting that synaptotagmin is not required for axonogenesis in the CNS or PNS. Based on these findings, we suggest that the molecular mechanisms underlying vesicular-mediated membrane expansion during axonal outgrowth are distinct from those required for synaptic vesicle fusion during neurotransmitter release.

Synaptotagmin controls and modulates synaptic-vesicle fusion in a Ca(2+)-dependent manner.

Although numerous electrophysiological and biochemical studies have defined many of the properties of the putative Ca2+ receptor for exocytosis at the synapse, the molecular mechanisms that couple influx of Ca2+ and release of neurotransmitter have remained elusive. Several proteins have emerged recently as putative Ca2+ sensors. Interestingly, one of these proteins, synaptotagmin, shares many properties with the putative Ca2+ receptor. Recent genetic experiments in Caenorhabditis elegans, Drosophila and mouse have provided important insights about synaptotagmin's role in neurotransmitter release. These experiments, combined with electrophysiological and biochemical studies, suggest that synaptotagmin is a key Ca2+ sensor, converting the ubiquitously used cellular secretory pathway into a Ca(2+)-regulated exocytotic pathway.

Obstructed tracheal bronchus as a cause of post-obstructive pneumonia.

This article describes a case of obstructed supernumerary tracheal bronchus with partial atelectasis and pneumonia of the right upper lobe, diagnosed using trispiral tomograms. An obstructing broncholith, a supernumerary tracheal bronchus, and granulation tissue mass extruding from the bronchus were confirmed by resection of the pulmonary segment.

Presynaptic proteins involved in exocytosis in Drosophila melanogaster: a genetic analysis.

Neuronal communication involves the fusion of neurotransmitter filled synaptic vesicles with the presynaptic terminal. This exocytotic event depends upon proteins present in three separate compartments: the synaptic vesicle, the synaptic cytosol, and the presynaptic membrane. Recent data indicate that the basic components of exocytotic pathways, including those used for neurotransmitter release, are conserved from yeast to human. Genetic dissection of the secretory pathway in yeast, identification of the target proteins cleaved by the clostridial neurotoxins and biochemical characterization of the interactions of synaptic proteins from vertebrates have converged to provide the SNARE (soluble NSF attachment protein receptor) hypothesis for vesicle trafficking. This model proposes that proteins present in the vesicle (v-SNAREs) interact with membrane receptors (t-SNAREs) to provide a molecular scaffold for cytosolic proteins involved in fusion. The hypothesis that these mechanisms function at the synapse relies largely upon in vitro evidence. Recently, genetic approaches in mice, C. elegans and the fruitfly, Drosophila melanogaster, have been used to dissect the in vivo function of numerous proteins involved in synaptic transmission. This review covers recent progress and insights provided by a genetic dissection of neurotransmitter release in Drosophila. In addition, we will provide evidence that the mechanisms for synaptic communication are highly conserved from invertebrates to vertebrates, making Drosophila an ideal model system to further unravel the intricacies of synaptic transmission.