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SummaryThe exocyst is a complex of proteins originally identified in yeast that has been implicated in polarized secretion. Components of the exocyst have been implicated in neurite outgrowth, cell polarity, and cell viability. We have isolated an exocyst component, sec15, in a screen for genes required for synaptic specificity. Loss of sec15 causes a targeting defect of photoreceptors that coincides with mislocalization of specific cell adhesion and signaling molecules. Additionally, sec15 mutant neurons fail to localize other exocyst members like Sec5 and Sec8, but not Sec6, to neuronal terminals. However, loss of sec15 does not cause cell lethality in contrast to loss of sec5 or sec6. Our data suggest a role of Sec15 in an exocyst-like subcomplex for the targeting and subcellular distribution of specific proteins. The data also show that functions of other exocyst components persist in the absence of sec15, suggesting that different exocyst components have separable functions.

Active zones are highly specialized sites for release of neurotransmitter from presynaptic nerve terminals. The architecture of the active zone is exquisitely designed to facilitate the regulated tethering, docking, and fusing of the synaptic vesicles with the plasma membrane. Here we present our view of the structural and molecular organization of active zones across species and propose that all active zones are organized according to a common principle in which the structural differences correlate with the kinetics of transmitter release.

We describe the isolation and characterization of Drosophila synaptojanin (synj) mutants. synj encodes a phosphatidylinositol phosphatase involved in clathrin-mediated endocytosis. We show that Synj is specifically localized to presynaptic terminals and is associated with synaptic vesicles. The electrophysiological and ultrastructural defects observed in synj mutants are strikingly similar to those found in endophilin mutants, and Synj and Endo colocalize and interact biochemically. Moreover, synj; endo double mutant synaptic terminals exhibit properties that are very similar to terminals of each single mutant, and overexpression of Endophilin can partially rescue the functional defects in partial loss-of-function synj mutants. Interestingly, Synj is mislocalized and destabilized at synapses devoid of Endophilin, suggesting that Endophilin recruits and stabilizes Synj on newly formed vesicles to promote vesicle uncoating. Our data also provide further evidence that kiss-and-run is able to maintain neurotransmitter release when synapses are not extensively challenged.

The isolation of chemically induced mutations in forward genetic screens is one of the hallmarks of Drosophila genetics. However, mapping the corresponding loci and identifying the molecular lesions associated with these mutations are often difficult and labor-intensive. Two mapping methods are most often used in flies: meiotic recombination mapping with marked chromosomes and deficiency mapping. The availability of the fly genome sequence allows the establishment and usage of molecular markers. Single-nucleotide polymorphisms have therefore recently been used to map several genes. Here we show that thousands of molecularly mapped P element insertions in fly strains that are publicly available provide a powerful alternative method to single-nucleotide polymorphism mapping. We present a strategy that allows mapping of lethal mutations, as well as viable mutations with visible phenotypes, with minimal resources. The most important unknown in using recombination rates to map at high resolution is how accurately recombination data correlate with molecular maps in small intervals. We therefore surveyed distortions of recombination rates in intervals P elements in Drosophila.

Recent studies indicate that active zones (AZs)—sites of neurotransmitter release—may be assembled from preassembled AZ precursor vesicles inserted into the presynaptic plasma membrane. Here we report that one putative AZ precursor vesicle of CNS synapses—the Piccolo-Bassoon transport vesicle (PTV)—carries a comprehensive set of AZ proteins genetically and functionally coupled to synaptic vesicle exocytosis. Time-lapse imaging reveals that PTVs are highly mobile, consistent with a role in intracellular transport. Quantitative analysis reveals that the Bassoon, Piccolo, and RIM content of individual PTVs is, on average, half of that of individual presynaptic boutons and shows that the synaptic content of these molecules can be quantitatively accounted for by incorporation of integer numbers (typically two to three) of PTVs into presynaptic membranes. These findings suggest that AZs are assembled from unitary amounts of AZ material carried on PTVs.

Axon degeneration is a common and often early feature of neurodegeneration that correlates with the clinical manifestations and progression of neurological disease. Nicotinamide mononucleotide adenylytransferase (NMNAT) is a neuroprotective factor that delays axon degeneration following injury and in models of neurodegenerative diseases suggesting a converging molecular pathway of axon self-destruction. The underlying mechanisms have been under intense investigation and recent reports suggest a central role for axonal mitochondria in both degeneration and NMNAT/WLD(S) (Wallerian degeneration slow)-mediated protection. We used dorsal root ganglia (DRG) explants and Drosophila larval motor neurons (MNs) as models to address the role of mitochondria in Wallerian degeneration (WD). We find that expression of Drosophila NMNAT delays WD in human DRG neurons demonstrating evolutionary conservation of NMNAT function. Morphological comparison of mitochondria from WLD(S)-protected axons demonstrates that mitochondria shrink post-axotomy, though analysis of complex IV activity suggests that they retain their functional capacity despite this morphological change. To determine whether mitochondria are a critical site of regulation for WD, we genetically ablated mitochondria from Drosophila MN axons via the mitochondria trafficking protein milton. Milton loss-of-function did not induce axon degeneration in Drosophila larval MNs, and when axotomized WD proceeded stereotypically in milton distal axons although with a mild, but significant delay. Remarkably, the protective effects of NMNAT/WLD(S) were also maintained in axons devoid of mitochondria. These experiments unveil an axon self-destruction cascade governing WD that is not initiated by axonal mitochondria and for the first time illuminate a mitochondria-independent mechanism(s) regulating WD and NMNAT/WLD(S)-mediated axon protection.

Active zones are specialized presynaptic structures critical for neurotransmission. We show that a neuronal maintenance factor, nicotinamide mononucleotide adenylyltransferase (NMNAT), is required for maintaining active zone structural integrity in Drosophila by interacting with the active zone protein, Bruchpilot (BRP), and shielding it from activity-induced ubiquitin–proteasome-mediated degradation. NMNAT localizes to the peri-active zone and interacts biochemically with BRP in an activity-dependent manner. Loss of NMNAT results in ubiquitination, mislocalization and aggregation of BRP, and subsequent active zone degeneration. We propose that, as a neuronal maintenance factor, NMNAT specifically maintains active zone structure by direct protein–protein interaction.

The selective degeneration of dendrites precedes neuronal cell death in hypoxia-ischemia (HI) and is a neuropathological hallmark of stroke. While it is clear that a number of different molecular pathways likely contribute to neuronal cell death in HI, the mechanisms that govern HI-induced dendrite degeneration are largely unknown. Here, we show that the NAD synthase nicotinamide mononucleotide adenylyltransferase (Nmnat) functions endogenously to protect Drosophila class IV dendritic arborization (da) sensory neurons against hypoxia-induced dendritic damage. Whereas dendrites of wild-type class IV neurons are largely resistant to morphological changes during prolonged periods of hypoxia (

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Dendrites can be maintained for extended periods of time after they initially establish coverage of their receptive field. The long-term maintenance of dendrites underlies synaptic connectivity, but how neurons establish and then maintain their dendritic arborization patterns throughout development is not well understood. Here, we show that the NAD synthase Nicotinamide mononucleotide adenylyltransferase (Nmnat) is cell-autonomously required for maintaining type-specific dendritic coverage of Drosophila dendritic arborization (da) sensory neurons. In nmnat heterozygous mutants, dendritic arborization patterns of class IV da neurons are properly established before increased retraction and decreased growth of terminal branches lead to progressive defects in dendritic coverage during later stages of development. Although sensory axons are largely intact in nmnat heterozygotes, complete loss of nmnat function causes severe axonal degeneration, demonstrating differential requirements for nmnat dosage in the maintenance of dendritic arborization patterns and axonal integrity. Overexpression of Nmnat suppresses dendrite maintenance defects associated with loss of the tumor suppressor kinase Warts (Wts), providing evidence that Nmnat, in addition to its neuroprotective role in axons, can function as a protective factor against progressive dendritic loss. Moreover, motor neurons deficient for nmnat show progressive defects in both dendrites and axons. Our studies reveal an essential role for endogenous Nmnat function in the maintenance of both axonal and dendritic integrity and present evidence of a broad neuroprotective role for Nmnat in the central nervous system.

Neurodegeneration can be triggered by genetic or environmental factors. Although the precise cause is often unknown, many neurodegenerative diseases share common features such as protein aggregation and age dependence. Recent studies in Drosophila have uncovered protective effects of NAD synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) against activity-induced neurodegeneration and injury-induced axonal degeneration1,2. Here we show that NMNAT overexpression can also protect against spinocerebellar ataxia 1 (SCA1)-induced neurodegeneration, suggesting a general neuroprotective function of NMNAT. It protects against neurodegeneration partly through a proteasome-mediated pathway in a manner similar to heat-shock protein 70 (Hsp70). NMNAT displays chaperone function both in biochemical assays and cultured cells, and it shares significant structural similarity with known chaperones. Furthermore, it is upregulated in the brain upon overexpression of poly-glutamine expanded protein and recruited with the chaperone Hsp70 into protein aggregates. Our results implicate NMNAT as a stress-response protein that acts as a chaperone for neuronal maintenance and protection. Our studies provide an entry point for understanding how normal neurons maintain activity, and offer clues for the common mechanisms underlying different neurodegenerative conditions.

The architecture of the presynaptic release site is exquisitely designed to facilitate the regulated tethering, docking, and fusing of synaptic vesicles with the plasma membrane. With the identification of some of the building blocks, we are beginning to understand the morphologic and functional properties of the synapse. Presynaptic release sites consist of a plasma membrane, a cytomatrix, and dense projections. These three components are morphologically distinct, yet they are intimately connected with each other and the postsynaptic nerve terminal, ensuring the fidelity of synaptic vesicle tethering, docking and fusion, as well as signal detection. Although the morphology of active zones and the molecular composition vary among species, tissues and cells, the architectural design of the release sites is likely conserved.

Wallerian degeneration refers to a loss of the distal part of an axon after nerve injury. Wallerian degeneration slow (Wlds) mice overexpress a chimeric protein containing the NAD synthase NMNAT (nicotinamide mononucleotide adenylyltransferase 1) and exhibit a delay in axonal degeneration. Currently, conflicting evidence raises questions as to whether NMNAT is the protecting factor and whether its enzymatic activity is required for such a possible function. Importantly, the link between nmnat and axon degeneration is at present solely based on overexpression studies of enzymatically active protein. Here we use the visual system of Drosophila as a model system to address these issues. We have isolated the first nmnat mutations in a multicellular organism in a forward genetic screen for synapse malfunction in Drosophila. Loss of nmnat causes a rapid and severe neurodegeneration that can be attenuated by blocking neuronal activity. Furthermore, in vivo neuronal expression of mutated nmnat shows that enzymatically inactive NMNAT protein retains strong neuroprotective effects and rescues the degeneration phenotype caused by loss of nmnat. Our data indicate an NAD-independent requirement of NMNAT for maintaining neuronal integrity that can be exploited to protect neurons from neuronal activity-induced degeneration by overexpression of the protein., The first mutant analysis of NMNAT (nicotinamide mononucleotide adenylyltransferase 1) reveals an essential neuronal protective role that functions independently of NMNAT's enzymatic activity. NMNAT can also be exploited to protect neurons against activity-induced neurodegeneration.

Summary Specifying synaptic partners and regulating synaptic numbers are at least partly activity-dependent processes during visual map formation in all systems investigated to date [1–5]. In Drosophila, six photoreceptors that view the same point in visual space have to be sorted into synaptic modules called cartridges in order to form a visuotopically correct map [6, 7]. Synapse numbers per photoreceptor terminal and cartridge are both precisely regulated [8–10]. However, it is unknown whether an activity-dependent mechanism or a genetically encoded developmental program regulates synapse numbers. We performed a large-scale quantitative ultrastructural analysis of photoreceptor synapses in mutants affecting the generation of electrical potentials ( norpA , trp;trpl ), neurotransmitter release ( hdc , syt ), vesicle endocytosis ( synj ), the trafficking of specific guidance molecules during photoreceptor targeting ( sec15 ), a specific guidance receptor required for visual map formation ( Dlar ), and 57 other novel synaptic mutants affecting 43 genes. Remarkably, in all these mutants, individual photoreceptors form the correct number of synapses per presynaptic terminal independently of cartridge composition. Hence, our data show that each photoreceptor forms a precise and constant number of afferent synapses independently of neuronal activity and partner accuracy. Our data suggest cell-autonomous control of synapse numbers as part of a developmental program of activity-independent steps that lead to a "hard-wired" visual map in the fly brain.

SummaryThe V0 complex forms the proteolipid pore of an ATPase that acidifies vesicles. In addition, an independent function in membrane fusion has been proposed largely based on yeast vacuolar fusion experiments. We have isolated mutations in the largest V0 component vha100-1 in flies in an unbiased genetic screen for synaptic malfunction. The protein is only required in neurons, colocalizes with markers for synaptic vesicles as well as active zones, and interacts with t-SNAREs. Loss of vha100-1 leads to vesicle accumulation in synaptic terminals, suggesting a deficit in release. The amplitude of spontaneous release events and release with hypertonic stimulation indicate normal levels of neurotransmitter loading, yet mutant embryos display severe defects in evoked synaptic transmission and FM1-43 uptake. Our data suggest that Vha100-1 functions downstream of SNAREs in synaptic vesicle fusion.

Synapses of the mammalian CNS are asymmetric sites of cell–cell adhesion between nerve cells. They are designed to mediate the rapid and efficient transmission of signals from the presynaptic bouton of one neuron to the postsynaptic plasma membrane of a second neuron. Significant progress has been made in the characterization of the structural, functional and developmental assembly of CNS synapses. Recent progress has been made in understanding the molecular and cellular mechanisms that underlie synaptogenesis, in particular that of glutamatergic synapses of the CNS.

The innate immune response provides the first line of defense against pathogens by responding to foreign molecules, such as cytosolic DNA or double-stranded RNA, by-products of bacterial and viral infections. The mammalian protein STING (STimulator of INterferon Genes), an intracellular sensor to by-products of pathogenic infection, is critical to the innate immune response and the induction of interferon beta, a potent antimicrobial molecule. In Drosophila, fat body cells contain surface receptors for the detection of pathogens in the hemolymph. Activation of these receptors stimulates the innate immune response, which is potentiated by the induction of antimicrobial peptides. While the mechanisms of cell surface receptor signaling in Drosophila are well understood, less is known about the intracellular sensors to pathogenic infection. Here we describe the identification of a Drosophila homologue of human STING, dSTING. We show that dSTING is a transmembrane protein that localizes to the endoplasmic reticulum. dSTING is capable of binding nucleic acids, which results in its perinuclear translocation, presumably necessary for the induction of antimicrobial peptides. In both S2 cells and adult flies, the loss of dSTING by RNA interference results in increased pathogen replication and mortality, correlated to decreased antimicrobial induction. Functional genomics analysis shows that the loss of dSTING results in the inability of flies to mount an innate immune response upon pathogenic infection. These results suggest that dSTING is the evolutionary ancestor of cytosolic pathogen-associated nucleic acid-sensing, responsible for a proper immune response to infection.

Microtubule-mediated transport is essential for neuronal viability, neurite extension, synapse formation and synaptic function. Now a new kinesin receptor, syntabulin, has been identified that controls transport of the t-SNARE syntaxin along microtubules.