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Kendal Broadie

Publications and source records attributed to Kendal Broadie.

At least 19 recordsLinked to original sources
PubMed2006

Rolling blackout is required for synaptic vesicle exocytosis.

Rolling blackout (RBO) is a putative transmembrane lipase required for phospholipase C-dependent phosphatidylinositol 4,5-bisphosphate-diacylglycerol signaling in Drosophila neurons. Conditional temperature-sensitive (TS) rbo mutants display complete, reversible paralysis within minutes, demonstrating that RBO is acutely required for movement. RBO protein is localized predominantly in presynaptic boutons at neuromuscular junction (NMJ) synapses and throughout central synaptic neuropil, and rbo TS mutants display a complete, reversible block of both central and peripheral synaptic transmission within minutes. This phenotype appears limited to adults, because larval NMJs do not manifest the acute blockade. Electron microscopy of adult rbo TS mutant boutons reveals an increase in total synaptic vesicle (SV) content, with a concomitant shrinkage of presynaptic bouton size and an accumulation of docked SVs at presynaptic active zones within minutes. Genetic tests reveal a synergistic interaction between rbo and syntaxin1A TS mutants, suggesting that RBO is required in the mechanism of N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated SV exocytosis, or in a parallel pathway necessary for SV fusion. The rbo TS mutation does not detectably alter SNARE complex assembly, suggesting a downstream requirement in SV fusion. We conclude that RBO plays an essential role in neurotransmitter release, downstream of SV docking, likely mediating SV fusion.

Animals↗
PubMed2005

An essential Drosophila glutamate receptor subunit that functions in both central neuropil and neuromuscular junction.

A Drosophila forward genetic screen for mutants with defective synaptic development identified bad reception (brec). Homozygous brec mutants are embryonic lethal, paralyzed, and show no detectable synaptic transmission at the glutamatergic neuromuscular junction (NMJ). Genetic mapping, complementation tests, and genomic sequencing show that brec mutations disrupt a previously uncharacterized ionotropic glutamate receptor subunit, named here "GluRIID." GluRIID is expressed in the postsynaptic domain of the NMJ, as well as widely throughout the synaptic neuropil of the CNS. In the NMJ of null brec mutants, all known glutamate receptor subunits are undetectable by immunocytochemistry, and all functional glutamate receptors are eliminated. Thus, we conclude that GluRIID is essential for the assembly and/or stabilization of glutamate receptors in the NMJ. In null brec mutant embryos, the frequency of periodic excitatory currents in motor neurons is significantly reduced, demonstrating that CNS motor pattern activity is regulated by GluRIID. Although synaptic development and molecular differentiation appear otherwise unperturbed in null mutants, viable hypomorphic brec mutants display dramatically undergrown NMJs by the end of larval development, suggesting that GluRIID-dependent central pattern activity regulates peripheral synaptic growth. These studies reveal GluRIID as a newly identified glutamate receptor subunit that is essential for glutamate receptor assembly/stabilization in the peripheral NMJ and required for properly patterned motor output in the CNS.

Animals↗
PubMed2005

Protein expression profiling of the drosophila fragile X mutant brain reveals up-regulation of monoamine synthesis.

Fragile X syndrome is the most common form of inherited mental retardation, associated with both cognitive and behavioral anomalies. The disease is caused by silencing of the fragile X mental retardation 1 (fmr1) gene, which encodes the mRNA-binding, translational regulator FMRP. Previously we established a disease model through mutation of Drosophila fmr1 (dfmr1) and showed that loss of dFMRP causes defects in neuronal structure, function, and behavioral output similar to the human disease state. To uncover molecular targets of dFMRP in the brain, we use here a proteomic approach involving two-dimensional difference gel electrophoresis analyses followed by mass spectrometry identification of proteins with significantly altered expression in dfmr1 null mutants. We then focus on two misregulated enzymes, phenylalanine hydroxylase (Henna) and GTP cyclohydrolase (Punch), both of which mediate in concert the synthetic pathways of two key monoamine neuromodulators, dopamine and serotonin. Brain enzymatic assays show a nearly 2-fold elevation of Punch activity in dfmr1 null mutants. Consistently brain neurochemical assays show that both dopamine and serotonin are significantly increased in dfmr1 null mutants. At a cellular level, dfmr1 null mutant neurons display a highly significant elevation of the dense core vesicles that package these monoamine neuromodulators for secretion. Taken together, these data indicate that dFMRP normally down-regulates the monoamine pathway, which is consequently up-regulated in the mutant condition. Elevated brain levels of dopamine and serotonin provide a plausible mechanistic explanation for aspects of cognitive and behavioral deficits in human patients.

Animals↗
PubMed2005

Fathoming fragile X in fruit flies.

Fragile X syndrome (FraX) is the most common inherited mental retardation disease. It is caused by mutation of the fragile X mental retardation 1 (fmr1) gene. The FMR1 protein (FMRP) is a widely expressed RNA-binding translational regulator with reportedly hundreds of potential targets. Recent work has focused on putative roles of FMRP in regulating the development and plasticity of neuronal synaptic connections. The newest animal model of FraX, the fruit fly Drosophila, has revealed several novel mechanistic insights into the disease. This review focuses on Drosophila FMRP as (i) a negative regulator of translation via noncoding RNA, including microRNA and adaptor BC1 RNA-mediated silencing mechanisms; (ii) a negative regulator of microtubule cytoskeleton stability; and (iii) a negative regulator of neuronal architectural complexity.

Animals↗
PubMed2005

Lipid regulation of the synaptic vesicle cycle.

Membrane vesicle cycling is orchestrated through the combined actions of proteins and lipids. At neuronal synapses, this orchestration must meet the stringent demands of speed, fidelity and sustainability of the synaptic vesicle cycle that mediates neurotransmission. Historically, the lion's share of the attention has been focused on the proteins that are involved in this cycle; but, in recent years, it has become clear that the previously unheralded plasma membrane and vesicle lipids are also key regulators of this cycle. This article reviews recent insights into the roles of lipid-modifying enzymes and lipids in the acute modulation of neurotransmission.

Animals↗
PubMed2004

The Drosophila fragile X gene negatively regulates neuronal elaboration and synaptic differentiation.

Fragile X Syndrome (FraX) is the most common form of inherited mental retardation. The disease is caused by the silencing of the fragile X mental retardation 1 (fmr1) gene, which encodes the RNA binding translational regulator FMRP . In FraX patients and fmr1 knockout mice, loss of FMRP causes denser and morphologically altered postsynaptic dendritic spines . Previously, we established a Drosophila FraX model and showed that dFMRP acts as a negative translational regulator of Futsch/MAP1B and negatively regulates synaptic branching and structural elaboration in the peripheral neuromuscular junction (NMJ) . Here, we investigate the role of dFMRP in the central brain, focusing on the mushroom body (MB), the learning and memory center . In MB neurons, dFMRP bidirectionally regulates multiple levels of structural architecture, including process formation from the soma, dendritic elaboration, axonal branching, and synaptogenesis. Drosophila fmr1 (dfmr) null mutant neurons display more complex architecture, including overgrowth, overbranching, and abnormal synapse formation. In contrast, dFMRP overexpression simplifies neuronal structure, causing undergrowth, underbranching, and loss of synapse differentiation. Studies of ultrastructural dfmr mutant neurons reveal enlarged and irregular synaptic boutons with dense accumulation of synaptic vesicles. Taken together, these data show that dFMRP is a potent negative regulator of neuronal architecture and synaptic differentiation in both peripheral and central nervous systems.

Animals↗
PubMed2004

The Drosophila metabotropic glutamate receptor DmGluRA regulates activity-dependent synaptic facilitation and fine synaptic morphology.

In vertebrates, several groups of metabotropic glutamate receptors (mGluRs) are known to modulate synaptic properties. In contrast, the Drosophila genome encodes a single functional mGluR (DmGluRA), an ortholog of vertebrate group II mGluRs, greatly expediting the functional characterization of mGluR-mediated signaling in the nervous system. We show here that DmGluRA is expressed at the glutamatergic neuromuscular junction (NMJ), localized in periactive zones of presynaptic boutons but excluded from active sites. Null DmGluRA mutants are completely viable, and all of the basal NMJ synaptic transmission properties are normal. In contrast, DmGluRA mutants display approximately a threefold increase in synaptic facilitation during short stimulus trains. Prolonged stimulus trains result in very strongly increased ( approximately 10-fold) augmentation, including the appearance of asynchronous, bursting excitatory currents never observed in wild type. Both defects are rescued by expression of DmGluRA only in the neurons, indicating a specific presynaptic requirement. These phenotypes are reminiscent of hyperexcitable mutants, suggesting a role of DmGluRA signaling in the regulation of presynaptic excitability properties. The mutant phenotypes could not be replicated by acute application of mGluR antagonists, suggesting that DmGluRA regulates the development of presynaptic properties rather than directly controlling short-term modulation. DmGluRA mutants also display mild defects in NMJ architecture: a decreased number of synaptic boutons accompanied by an increase in mean bouton size. These morphological changes bidirectionally correlate with DmGluRA levels in the presynaptic terminal. These data reveal the following two roles for DmGluRA in presynaptic mechanisms: (1) modulation of presynaptic excitability properties important for the control of activity-dependent neurotransmitter release and (2) modulation of synaptic architecture.

Action Potentials↗
PubMed2004

Synapse scaffolding: intersection of endocytosis and growth.

The Drosophila dynamin-associated protein Dap160, homolog of the vertebrate Intersectins, is thought likely to act as a molecular scaffold in the synaptic periactive zone. New mutant analyses have revealed separable roles for Dap160 in the regulation of vesicular endocytosis and synaptic growth.

Adaptor Proteins, Vesicular Transport↗
PubMed2004

Rolling blackout, a newly identified PIP2-DAG pathway lipase required for Drosophila phototransduction.

The rolling blackout (rbo) gene encodes an integral plasma membrane lipase required for Drosophila phototransduction. Photoreceptors are enriched for the RBO protein, and temperature-sensitive rbo mutants show reversible elimination of phototransduction within minutes, demonstrating an acute requirement for the protein. The block is activity dependent, indicating that the action of RBO is use dependent. Conditional rbo mutants show activity-dependent depletion of diacylglycerol and concomitant accumulation of phosphatidylinositol phosphate and phosphatidylinositol 4,5-bisphosphate within minutes of induction, suggesting rapid downregulation of phospholipase C (PLC) activity. The RBO requirement identifies an essential regulatory step in G-protein-coupled, PLC-dependent inositol lipid signaling mediating activation of TRP and TRPL channels during phototransduction.

Amino Acid Sequence↗
PubMed2004

Ceramidase regulates synaptic vesicle exocytosis and trafficking.

A screen for Drosophila synaptic dysfunction mutants identified slug-a-bed (slab). The slab gene encodes ceramidase, a central enzyme in sphingolipid metabolism and regulation. Sphingolipids are major constituents of lipid rafts, membrane domains with roles in vesicle trafficking, and signaling pathways. Null slab mutants arrest as fully developed embryos with severely reduced movement. The SLAB protein is widely expressed in different tissues but enriched in neurons at all stages of development. Targeted neuronal expression of slab rescues mutant lethality, demonstrating the essential neuronal function of the protein. C(5)-ceramide applied to living preparations is rapidly accumulated at neuromuscular junction (NMJ) synapses dependent on the SLAB expression level, indicating that synaptic sphingolipid trafficking and distribution is regulated by SLAB function. Evoked synaptic currents at slab mutant NMJs are reduced by 50-70%, whereas postsynaptic glutamate-gated currents are normal, demonstrating a specific presynaptic impairment. Hypertonic saline-evoked synaptic vesicle fusion is similarly impaired by 50-70%, demonstrating a loss of readily releasable vesicles. In addition, FM1-43 dye uptake is reduced in slab mutant presynaptic terminals, indicating a smaller cycling vesicle pool. Ultrastructural analyses of mutants reveal a normal vesicle distribution clustered and docked at active zones, but fewer vesicles in reserve regions, and a twofold to threefold increased incidence of vesicles linked together and tethered at the plasma membrane. These results indicate that SLAB ceramidase function controls presynaptic terminal sphingolipid composition to regulate vesicle fusion and trafficking, and thus the strength and reliability of synaptic transmission.

Animals↗
PubMed2004

Cellular bases of activity-dependent paralysis in Drosophila stress-sensitive mutants.

Stress-sensitive mutants in Drosophila have been shown to exhibit activity-dependent defects in neurotransmission. Using the neuromuscular junction (NMJ), this study investigates synaptic function more specifically in two stress-sensitive mutants: stress-sensitive B (sesB), which encodes a mitochondrial ADP/ATP translocase (ANT); and Atpalpha(2206), a conditional mutant of the Na+/K+ ATPase alpha-subunit. Mechanical shock induces a period of brief paralysis in both homozygous and double heterozygous mutants, but further analysis revealed distinct activity-dependent neurotransmission lesions in each mutant. Basal neurotransmission appeared similar to wild-type controls in both mutants under low frequency stimulation. High frequency stimulation, however, caused pronounced synaptic fatigue as well as slow and incomplete synaptic recovery in sesB mutants while Atpalpha(2206) mutants displayed an increase (25-fold) in synaptic failures. Perhaps to compensate for these activity dependent defects, the neuromuscular synapse was found to be overgrown in both mutants. Passive electrotonic stimulation, which initiates synaptic transmission independent of action potentials, ameliorated synaptic failures and resulted in increased neurotransmission amplitude in Atpalpha(2206) mutants. In addition, spontaneous synaptic vesicle fusion rates were increased in Atpalpha(2206) mutants, suggesting that, in the absence of action potential requirements, these synaptic terminals are healthy, if not hyperactive. Dye labeling studies revealed aberrant synaptic vesicle cycling in sesB mutants indicating a reduction of functional synaptic vesicles. We therefore postulate that both stress-sensitive mutants harbor unique neurotransmission defects: Atpalpha(2206) mutants are unable to maintain ionic gradients required during repetitive action potential propagation, and sesB mutants cannot maintain synaptic vesicle cycling during periods of high demand.

Animals↗
PubMed2004

The hereditary spastic paraplegia gene, spastin, regulates microtubule stability to modulate synaptic structure and function.

BACKGROUND: Hereditary Spastic Paraplegia (HSP) is a devastating neurological disease causing spastic weakness of the lower extremities and eventual axonal degeneration. Over 20 genes have been linked to HSP in humans; however, mutations in one gene, spastin (SPG4), are the cause of >40% of all cases. Spastin is a member of the ATPases associated with diverse cellular activities (AAA) protein family, and contains a microtubule interacting and organelle transport (MIT) domain. Previous work in cell culture has proposed a role for Spastin in regulating microtubules. RESULTS: Employing Drosophila transgenic methods for overexpression and RNA interference (RNAi), we have investigated the role of Spastin in vivo. We show that Drosophila Spastin (D-Spastin) is enriched in axons and synaptic connections. At neuromuscular junctions (NMJ), Dspastin RNAi causes morphological undergrowth and reduced synaptic area. Moreover, Dspastin overexpression reduces synaptic strength, whereas Dspastin RNAi elevates synaptic currents. By using antibodies against posttranslationally modified alpha-Tubulin, we find that Dspastin regulates microtubule stability. Functional synaptic defects caused by Dspastin RNAi and overexpression were pharmacologically alleviated by agents that destabilize and stabilize microtubules, respectively. CONCLUSIONS: Loss of Dspastin in Drosophila causes an aberrantly stabilized microtubule cytoskeleton in neurons and defects in synaptic growth and neurotransmission. These in vivo data strongly support previous reports, providing a probable cause for the neuronal dysfunction in spastin-linked HSP disease. The role of Spastin in regulating neuronal microtubule stability suggests therapeutic targets for HSP treatment and may provide insight into neurological disorders linked to microtubule dysfunction.

Animals↗
PubMed2004

The Drosophila fragile X-related gene regulates axoneme differentiation during spermatogenesis.

Macroorchidism (i.e., enlarged testicles) and mental retardation are the two hallmark symptoms of Fragile X syndrome (FraX). The disease is caused by loss of fragile X mental retardation protein (FMRP), an RNA-binding translational regulator. We previously established a FraX model in Drosophila, showing that the fly FMRP homologue, dFXR, acts as a negative translational regulator of microtubule-associated Futsch to control stability of the microtubule cytoskeleton during nervous system development. Here, we investigate dFXR function in the testes. Male dfxr null mutants have the enlarged testes characteristic of the disease and are nearly sterile (>90% reduced male fecundity). dFXR protein is highly enriched in Drosophila testes, particularly in spermatogenic cells during the early stages of spermatogenesis. Cytological analyses reveal that spermatogenesis is arrested specifically in late-stage spermatid differentiation following individualization. Ultrastructurally, dfxr mutants lose specifically the central pair microtubules in the sperm tail axoneme. The frequency of central pair microtubule loss becomes progressively greater as spermatogenesis progresses, suggesting that dFXR regulates microtubule stability. Proteomic analyses reveal that chaperones Hsp60B-, Hsp68-, Hsp90-related protein TRAP1, and other proteins have altered expression in dfxr mutant testes. Taken together with our previous nervous system results, these data suggest a common model in which dFXR regulates microtubule stability in both synaptogenesis in the nervous system and spermatogenesis in the testes. The characterization of dfxr function in the testes paves the way to genetic screens for modifiers of dfxr-induced male sterility, as a means to efficiently dissect FMRP-mediated mechanisms.

Animals↗
PubMed2003

The ubiquitin proteasome system acutely regulates presynaptic protein turnover and synaptic efficacy.

BACKGROUND: The ubiquitin proteasome system (UPS) mediates regulated protein degradation and provides a mechanism for closely controlling protein abundance in spatially restricted domains within cells. We hypothesized that the UPS may acutely determine the local concentration of key regulatory proteins at neuronal synapses as a means for locally modulating synaptic efficacy and the strength of neurotransmission communication. RESULTS: We investigated this hypothesis at the Drosophila neuromuscular synapse by using an array of genetic and pharmacological tools. This study demonstrates that UPS components are present in presynaptic boutons and that the UPS functions locally in the presynaptic compartment to rapidly eliminate a conditional transgenic reporter of proteasome activity. We assayed a panel of synaptic proteins to determine whether the UPS acutely regulates the local abundance of native synaptic targets. Both acute pharmacological inhibition of the proteasome (<1 hr) and targeted genetic perturbation of proteasome function in the presynaptic neuron cause the specific accumulation of the essential synaptic vesicle-priming protein DUNC-13. Most importantly, acute pharmacological inhibition of the proteasome (<1 hr) causes a rapid strengthening of neurotransmission (an approximately 50% increase in evoked amplitude) because of increased presynaptic efficacy. The proteasome-dependent regulation of presynaptic protein abundance, both of the exogenous reporter and native DUNC-13, and the modulation of presynaptic neurotransmitter release occur on an intermediate, rapid (tens of minutes) timescale. CONCLUSIONS: Taken together, these studies demonstrate that the UPS functions locally within synaptic boutons to acutely control levels of presynaptic protein and that the rate of UPS-dependent protein degradation is a primary determinant of neurotransmission strength.

Animals↗
PubMed2003

Synaptic Drosophila UNC-13 is regulated by antagonistic G-protein pathways via a proteasome-dependent degradation mechanism.

UNC-13 is a highly conserved plasma membrane-associated synaptic protein implicated in the regulation of neurotransmitter release through the direct modulation of the SNARE exocytosis complex. Previously, we characterized the Drosophila homologue (DUNC-13) and showed it to be essential for neurotransmitter release immediately upstream of vesicular fusion ("priming") at the neuromuscular junction (NMJ). Here, we show that the abundance of DUNC-13 in NMJ synaptic boutons is regulated downstream of GalphaS and Galphaq pathways, which have inhibitory and facilitatory roles, respectively. Both cAMP modulation and PKA function are required for DUNC-13 synaptic up-regulation, suggesting that the cAMP pathway enhances synaptic efficacy via DUNC-13. Similarly, PLC function and DAG modulation also regulate the synaptic levels of DUNC-13, through a mechanism that appears independent of PKC. Our results suggest that proteasome-mediated protein degradation is the primary mechanism regulating DUNC-13 levels at the synapse. Both PLC- and PKA-mediated pathways appear to regulate synaptic levels of DUNC-13 through controlling the rate of proteasome-dependent DUNC-13 degradation. We conclude that the functional abundance of DUNC-13 at the synapse, a key determinant of synaptic vesicle priming and neurotransmitter release probability, is primarily regulated by the rate of protein degradation, rather than translocation or transport, convergently controlled via both cAMP and DAG signal transduction pathways.

Acetylcysteine↗
PubMed2003

Mutation and activation of Galpha s similarly alters pre- and postsynaptic mechanisms modulating neurotransmission.

Constitutive activation of Galphas in the Drosophila brain abolishes associative learning, a behavioral disruption far worse than that observed in any single cAMP metabolic mutant, suggesting that Galphas is essential for synaptic plasticity. The intent of this study was to examine the role of Galphas in regulating synaptic function by targeting constitutively active Galphas to either pre- or postsynaptic cells and by examining loss-of-function Galphas mutants (dgs) at the glutamatergic neuromuscular junction (NMJ) model synapse. Surprisingly, both loss of Galphas and activation of Galphas in either pre- or postsynaptic compartment similarly increased basal neurotransmission, decreased short-term plasticity (facilitation and augmentation), and abolished posttetanic potentiation. Elevated synaptic function was specific to an evoked neurotransmission pathway because both spontaneous synaptic vesicle fusion frequency and amplitude were unaltered in all mutants. In the postsynaptic cell, the glutamate receptor field was regulated by Galphas activity; based on immunocytochemical studies, GluRIIA receptor subunits were dramatically downregulated (>75% decrease) in both loss and constitutive active Galphas genotypes. In the presynaptic cell, the synaptic vesicle cycle was regulated by Galphas activity; based on FM1-43 dye imaging studies, evoked vesicle fusion rate was increased in both loss and constitutively active Galphas genotypes. An important conclusion of this study is that both increased and decreased Galphas activity very similarly alters pre- and postsynaptic mechanisms. A second important conclusion is that Galphas activity induces transynaptic signaling; targeted Galphas activation in the presynapse downregulates postsynaptic GluRIIA receptors, whereas targeted Galphas activation in the postsynapse enhances presynaptic vesicle cycling.

Animals↗