Richard W. Daniels

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.

A dual leucine kinase–dependent axon self-destruction program promotes Wallerian degeneration

Axon degeneration underlies many common neurological disorders, but the signaling pathways that orchestrate axon degeneration are unknown. We found that dual leucine kinase (DLK) promoted degeneration of severed axons in Drosophila and mice, and that its target, c-Jun N-terminal kinase, promoted degeneration locally in axons as they committed to degenerate. This pathway also promoted degeneration after chemotherapy exposure and may be a component of a general axon self-destruction program.

A Single Vesicular Glutamate Transporter Is Sufficient to Fill a Synaptic Vesicle

Quantal size is the postsynaptic response to the release of a single synaptic vesicle and is determined in part by the amount of transmitter within that vesicle. At glutamatergic synapses, the vesicular glutamate transporter (VGLUT) fills vesicles with glutamate. While elevated VGLUT expression increases quantal size, the minimum number of transporters required to fill a vesicle is unknown. In Drosophila DVGLUT mutants, reduced transporter levels lead to a dose-dependent reduction in the frequency of spontaneous quantal release with no change in quantal size. Quantal frequency is not limited by vesicle number or impaired exocytosis. This suggests that a single functional unit of transporter is both necessary and sufficient to fill a vesicle to completion and that vesicles without DVGLUT are empty. Consistent with the presence of empty vesicles, at dvglut mutant synapses synaptic vesicles are smaller, suggesting that vesicle filling and/or transporter level is an important determinant of vesicle size.

Visualizing glutamatergic cell bodies and synapses in Drosophila larval and adult CNS

Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system (CNS) and at Drosophila neuromuscular junctions (NMJs). Although glutamate is also used as a transmitter in the Drosophila CNS, there has been no systematic description of the central glutamatergic signaling system in the fly. With the recent cloning of the Drosophila vesicular glutamate transporter (DVGLUT), it is now possible to mark many, if not all, central glutamatergic neurons and synapses. Here we present the pattern of glutamatergic synapses and cell bodies in the late larval CNS and in the adult fly brain by using an anti‐DVGLUT antibody. We also introduce two new tools for studying the Drosophila glutamatergic system: a dvglut promoter fragment fused to Gal4 whose expression labels glutamatergic neurons and a green fluorescent protein (GFP)‐tagged DVGLUT transgene that localizes to synapses. In the larval CNS, we find synaptic DVGLUT immunoreactivity prominent in all brain lobe neuropil compartments except for the mushroom body. Likewise in the adult CNS, glutamatergic synapses are abundant throughout all major brain structures except the mushroom body. We also find that the larval ventral nerve cord neuropil is rich in glutamatergic synapses, which are primarily located near the dorsal surface of the neuropil, segregated from the ventrally positioned cholinergic processes. This description of the glutamatergic system in Drosophila highlights the prevalence of glutamatergic neurons in the CNS and presents tools for future study and manipulation of glutamatergic transmission. J. Comp. Neurol. 508:131–152, 2008.

Rab3 Dynamically Controls Protein Composition at Active Zones

Synaptic transmission requires the localization of presynaptic release machinery to active zones. Mechanisms regulating the abundance of such synaptic proteins at individual release sites are likely determinants of site-specific synaptic efficacy. We now identify a role for the small GTPase Rab3 in regulating the distribution of presynaptic components to active zones. At Drosophila rab3 mutant NMJs, the presynaptic protein Bruchpilot, calcium channels, and electron-dense T bars are concentrated at a fraction of available active zones, leaving the majority of sites devoid of these key presynaptic release components. Late addition of Rab3 to mutant NMJs rapidly reverses this phenotype by recruiting Brp to sites previously lacking the protein, demonstrating that Rab3 can dynamically control the composition of the presynaptic release machinery. While previous studies of Rab3 have focused on its role in the synaptic vesicle cycle, these findings demonstrate an additional and unexpected function for Rab3 in the localization of presynaptic proteins to active zones.

DFsn collaborates with Highwire to down-regulate the Wallenda/DLK kinase and restrain synaptic terminal growth

BackgroundThe growth of new synapses shapes the initial formation and subsequent rearrangement of neural circuitry. Genetic studies have demonstrated that the ubiquitin ligase Highwire restrains synaptic terminal growth by down-regulating the MAP kinase kinase kinase Wallenda/dual leucine zipper kinase (DLK). To investigate the mechanism of Highwire action, we have identified DFsn as a binding partner of Highwire and characterized the roles of DFsn in synapse development, synaptic transmission, and the regulation of Wallenda/DLK kinase abundance.ResultsWe identified DFsn as an F-box protein that binds to the RING-domain ubiquitin ligase Highwire and that can localize to the Drosophila neuromuscular junction. Loss-of-function mutants for DFsn have a phenotype that is very similar to highwire mutants – there is a dramatic overgrowth of synaptic termini, with a large increase in the number of synaptic boutons and branches. In addition, synaptic transmission is impaired in DFsn mutants. Genetic interactions between DFsn and highwire mutants indicate that DFsn and Highwire collaborate to restrain synaptic terminal growth. Finally, DFsn regulates the levels of the Wallenda/DLK kinase, and wallenda is necessary for DFsn-dependent synaptic terminal overgrowth.ConclusionThe F-box protein DFsn binds the ubiquitin ligase Highwire and is required to down-regulate the levels of the Wallenda/DLK kinase and restrain synaptic terminal growth. We propose that DFsn and Highwire participate in an evolutionarily conserved ubiquitin ligase complex whose substrates regulate the structure and function of synapses.

The novel endosomal membrane protein Ema interacts with the class C Vps-HOPS complex to promote endosomal maturation.

Defective attenuation of BMP signaling causes synapses to overgrow in Drosophila Ema mutants due to impaired endosomal maturation.

Genetic modifiers of abnormal organelle biogenesis in a Drosophila model of BLOC-1 deficiency

Biogenesis of lysosome-related organelles complex 1 (BLOC-1) is a protein complex formed by the products of eight distinct genes. Loss-of-function mutations in two of these genes, DTNBP1 and BLOC1S3, cause Hermansky-Pudlak syndrome, a human disorder characterized by defective biogenesis of lysosome-related organelles. In addition, haplotype variants within the same two genes have been postulated to increase the risk of developing schizophrenia. However, the molecular function of BLOC-1 remains unknown. Here, we have generated a fly model of BLOC-1 deficiency. Mutant flies lacking the conserved Blos1 subunit displayed eye pigmentation defects due to abnormal pigment granules, which are lysosome-related organelles, as well as abnormal glutamatergic transmission and behavior. Epistatic analyses revealed that BLOC-1 function in pigment granule biogenesis requires the activities of BLOC-2 and a putative Rab guanine-nucleotide-exchange factor named Claret. The eye pigmentation phenotype was modified by misexpression of proteins involved in intracellular protein trafficking; in particular, the phenotype was partially ameliorated by Rab11 and strongly enhanced by the clathrin-disassembly factor, Auxilin. These observations validate Drosophila melanogaster as a powerful model for the study of BLOC-1 function and its interactions with modifier genes.

Drosophila Vesicular Monoamine Transporter Mutants Can Adapt to Reduced or Eliminated Vesicular Stores of Dopamine and Serotonin

Physiologic and pathogenic changes in amine release induce dramatic behavioral changes, but the underlying cellular mechanisms remain unclear. To investigate these adaptive processes, we have characterized mutations in the Drosophila vesicular monoamine transporter (dVMAT), which is required for the vesicular storage of dopamine, serotonin, and octopamine. dVMAT mutant larvae show reduced locomotion and decreased electrical activity in motoneurons innervating the neuromuscular junction (NMJ) implicating central amines in the regulation of these activities. A parallel increase in evoked glutamate release by the motoneuron is consistent with a homeostatic adaptation at the NMJ. Despite the importance of aminergic signaling for regulating locomotion and other behaviors, adult dVMAT homozygous null mutants survive under conditions of low population density, thus allowing a phenotypic characterization of adult behavior. Homozygous mutant females are sterile and show defects in both egg retention and development; males also show reduced fertility. Homozygotes show an increased attraction to light but are mildly impaired in geotaxis and escape behaviors. In contrast, heterozygous mutants show an exaggerated escape response. Both hetero- and homozygous mutants demonstrate an altered behavioral response to cocaine. dVMAT mutants define potentially adaptive responses to reduced or eliminated aminergic signaling and will be useful to identify the underlying molecular mechanisms.

Increased vesicular glutamate transporter expression causes excitotoxic neurodegeneration.

Increases in vesicular glutamate transporter (VGLUT) levels are observed after a variety of insults including hypoxic injury, stress, methamphetamine treatment, and in genetic seizure models. Such overexpression can cause an increase in the amount of glutamate released from each vesicle, but it is unknown whether this is sufficient to induce excitotoxic neurodegeneration. Here we show that overexpression of the Drosophila vesicular glutamate transporter (DVGLUT) leads to excess glutamate release, with some vesicles releasing several times the normal amount of glutamate. Increased DVGLUT expression also leads to an age-dependent loss of motor function and shortened lifespan, accompanied by a progressive neurodegeneration in the postsynaptic targets of the DVGLUT-overexpressing neurons. The early onset lethality, behavioral deficits, and neuronal pathology require overexpression of a functional DVGLUT transgene. Thus overexpression of DVGLUT is sufficient to generate excitotoxic neuropathological phenotypes and therefore reducing VGLUT levels after nervous system injury or stress may mitigate further damage.