Aaron DiAntonio

Genetic Analysis of Glutamate Receptors in Drosophila Reveals a Retrograde Signal Regulating Presynaptic Transmitter Release

Postsynaptic sensitivity to glutamate was genetically manipulated at the Drosophila neuromuscular junction (NMJ) to test whether postsynaptic activity can regulate presynaptic function during development. We cloned the gene encoding a second muscle-specific glutamate receptor, DGluRIIB, which is closely related to the previously identified DGluRIIA and located adjacent to it in the genome. Mutations that eliminate DGluRIIA (but not DGluRIIB) or transgenic constructs that increase DGluRIIA expression were generated. When DGluRIIA is missing, the response of the muscle to a single vesicle of transmitter is substantially decreased. However, the response of the muscle to nerve stimulation is normal because quantal content is significantly increased. Thus, a decrease in postsynaptic receptors leads to an increase in presynaptic transmitter release, indicating that postsynaptic activity controls a retrograde signal that regulates presynaptic function.

Ubiquitination-dependent mechanisms regulate synaptic growth and function

The covalent attachment of ubiquitin to cellular proteins is a powerful mechanism for controlling protein activity and localization. Ubiquitination is a reversible modification promoted by ubiquitin ligases and antagonized by deubiquitinating proteases. Ubiquitin-dependent mechanisms regulate many important processes including cell-cycle progression, apoptosis and transcriptional regulation. Here we show that ubiquitin-dependent mechanisms regulate synaptic development at the Drosophila neuromuscular junction (NMJ). Neuronal overexpression of the deubiquitinating protease fat facets leads to a profound disruption of synaptic growth control; there is a large increase in the number of synaptic boutons, an elaboration of the synaptic branching pattern, and a disruption of synaptic function. Antagonizing the ubiquitination pathway in neurons by expression of the yeast deubiquitinating protease UBP2 (ref. 5) also produces synaptic overgrowth and dysfunction. Genetic interactions between fat facets and highwire, a negative regulator of synaptic growth that has structural homology to a family of ubiquitin ligases, suggest that synaptic development may be controlled by the balance between positive and negative regulators of ubiquitination.

Highwire regulates synaptic growth in Drosophila.

The formation, stabilization, and growth of synaptic connections are dynamic and highly regulated processes. The glutamatergic neuromuscular junction (NMJ) in Drosophila grows new boutons and branches throughout larval development. A primary walking behavior screen followed by a secondary anatomical screen led to the identification of the highwire (hiw) gene. In hiw mutants, the specificity of motor axon pathfinding and synapse formation appears normal. However, NMJ synapses grow exuberantly and are greatly expanded in both the number of boutons and the extent and length of branches. These synapses appear normal ultrastructurally but have reduced quantal content physiologically. hiw encodes a large protein found at presynaptic terminals. Within presynaptic terminals, HIW is localized to the periactive zone surrounding active zones; Fasciclin II (Fas II), which also controls synaptic growth, is found at the same location.

Highwire Restrains Synaptic Growth by Attenuating a MAP Kinase Signal

Highwire is an extremely large, evolutionarily conserved E3 ubiquitin ligase that negatively regulates synaptic growth at the Drosophila NMJ. Highwire has been proposed to restrain synaptic growth by downregulating a synaptogenic signal. Here we identify such a downstream signaling pathway. A screen for suppressors of the highwire synaptic overgrowth phenotype yielded mutations in wallenda, a MAP kinase kinase kinase (MAPKKK) homologous to vertebrate DLK and LZK. wallenda is both necessary for highwire synaptic overgrowth and sufficient to promote synaptic overgrowth, and synaptic levels of Wallenda protein are controlled by Highwire and ubiquitin hydrolases. highwire synaptic overgrowth requires the MAP kinase JNK and the transcription factor Fos. These results suggest that Highwire controls structural plasticity of the synapse by regulating gene expression through a MAP kinase signaling pathway. In addition to controlling synaptic growth, Highwire promotes synaptic function through a separate pathway that does not require wallenda.

Increased Expression of the Drosophila Vesicular Glutamate Transporter Leads to Excess Glutamate Release and a Compensatory Decrease in Quantal Content

Quantal size is a fundamental parameter controlling the strength of synaptic transmission. The transmitter content of synaptic vesicles is one mechanism that can affect the physiological response to the release of a single vesicle. At glutamatergic synapses, vesicular glutamate transporters (VGLUTs) are responsible for filling synaptic vesicles with glutamate. To investigate how VGLUT expression can regulate synaptic strength in vivo, we have identified the Drosophila vesicular glutamate transporter, which we name DVGLUT. DVGLUT mRNA is expressed in glutamatergic motoneurons and a large number of interneurons in the Drosophila CNS. DVGLUT protein resides on synaptic vesicles and localizes to the presynaptic terminals of all known glutamatergic neuromuscular junctions as well as to synapses throughout the CNS neuropil. Increasing the expression of DVGLUT in motoneurons leads to an increase in quantal size that is accompanied by an increase in synaptic vesicle volume. At synapses confronted with increased glutamate release from each vesicle, there is a compensatory decrease in the number of synaptic vesicles released that maintains normal levels of synaptic excitation. These results demonstrate that (1) expression of DVGLUT determines the size and glutamate content of synaptic vesicles and (2) homeostatic mechanisms exist to attenuate the excitatory effects of excess glutamate release.

Differential Localization of Glutamate Receptor Subunits at the Drosophila Neuromuscular Junction

The subunit composition of postsynaptic neurotransmitter receptors is a key determinant of synaptic physiology. Two glutamate receptor subunits, Drosophila glutamate receptor IIA (DGluRIIA) and DGluRIIB, are expressed at the Drosophila neuromuscular junction and are redundant for viability, yet differ in their physiological properties. We now identify a third glutamate receptor subunit at the Drosophila neuromuscular junction, DGluRIII, which is essential for viability. DGluRIII is required for the synaptic localization of DGluRIIA and DGluRIIB and for synaptic transmission. Either DGluRIIA or DGluRIIB, but not both, is required for the synaptic localization of DGluRIII. DGluRIIA and DGluRIIB compete with each other for access to DGluRIII and subsequent localization to the synapse. These results are consistent with a model of a multimeric receptor in which DGluRIII is an essential component. At single postsynaptic cells that receive innervation from multiple motoneurons, DGluRIII is abundant at all synapses. However, DGluRIIA and DGluRIIB are differentially localized at the postsynaptic density opposite distinct motoneurons. Hence, innervating motoneurons may regulate the subunit composition of their receptor fields within a shared postsynaptic cell. The capacity of presynaptic inputs to shape the subunit composition of postsynaptic receptors could be an important mechanism for synapse-specific regulation of synaptic function and plasticity.

Glutamate Receptor Expression Regulates Quantal Size and Quantal Content at the Drosophila Neuromuscular Junction

At the Drosophila glutamatergic neuromuscular junction, the postsynaptic cell can regulate synaptic strength by both changing its sensitivity to neurotransmitter and generating a retrograde signal that regulates presynaptic transmitter release. To investigate the molecular mechanisms underlying these forms of plasticity, we have undertaken a genetic analysis of two postsynaptic glutamate receptors that are expressed at this synapse. Deletion of both genes results in embryonic lethality that can be rescued by transgenic expression of either receptor. Although these receptors are redundant for viability, they have important differences. By transgenically rescuing the double mutant, we have investigated the relationship of receptor gene dosage and composition to synaptic function. We find that the receptor subunit composition regulates quantal size, Argiotoxin sensitivity, and receptor desensitization kinetics. Finally, we show that the activity of the receptor can regulate the retrograde signal functioning at this synapse. Thus, the diversity of receptors expressed at this synapse provides the cell with mechanisms for generating synaptic plasticity.

Synaptic transmission persists in synaptotagmin mutants of Drosophila.

Synaptotagmin is one of the major integral membrane proteins of synaptic vesicles. It has been postulated to dock vesicles to their release sites, to act as the Ca2+ sensor for the release process, and to be a fusion protein during exocytosis. To clarify the function of this protein, we have undertaken a genetic analysis of the synaptotagmin gene in Drosophila. We have identified five lethal alleles of synaptotagmin, at least one of which lacks detectable protein. Surprisingly, however, many embryos homozygous for this null allele hatch and, as larvae, crawl, feed, and respond to stimuli. Electrophysiological recordings in embryonic cultures confirmed that synaptic transmission persists in the null allele. Therefore, synaptotagmin is not absolutely required for the regulated exocytosis of synaptic vesicles. The lethality of synaptotagmin in late first instar larvae is probably due to a perturbation of transmission that leaves the main apparatus for vesicle docking and fusion intact.

The effect on synaptic physiology of synaptotagmin mutations in drosophila

Synaptotagmin is a synaptic vesicle protein implicated in neurotransmitter release. Molecular characterization of four mutant alleles of this protein in Drosophila melanogaster has permitted an investigation of synaptotagmins role in synaptic physiology and of some of the structural requirements for its function. Reduced levels of synaptotagmin resulted in a substantial alteration in synaptic function in the eye and at larval neuromuscular junctions. Decreased neurotransmitter release caused smaller evoked synaptic potentials. However, the frequency, but not the size, of spontaneous quantal events was simultaneously increased. These abnormalities do not appear to be secondary to a detectable morphological change in the arborization of the synapse. The increased frequency of spontaneous events was insufficient to deplete significantly the vesicle supply and thereby account for reduced transmission. These data are discussed in the context of models in which synaptotagmins function includes a role in vesicle docking.

Synaptic development: insights from Drosophila

In Drosophila, the larval neuromuscular junction is particularly tractable for studying how synapses develop and function. In contrast to vertebrate central synapses, each presynaptic motor neuron and postsynaptic muscle cell is unique and identifiable, and the wiring circuit is invariant. Thus, the full power of Drosophila genetics can be brought to bear on a single, reproducibly identifiable, synaptic terminal. Each individual neuromuscular junction encompasses hundreds of synaptic neurotransmitter release sites housed in a chain of synaptic boutons. Recent advances have increased our understanding of the mechanisms that shape the development of both individual synapses--that is, the transmitter release sites including active zones and their apposed glutamate receptor clusters--and the whole synaptic terminal that connects a pre- and post-synaptic cell.