PubMed Health⌕ Search

Biomedical subjects

J Troy Littleton

Publications and source records attributed to J Troy Littleton.

14 recordsLinked to original sources
PubMed2006

Mixing and matching during synaptic vesicle endocytosis.

The question of how synapses maintain an active recycling pool of synaptic vesicles to support high-frequency synaptic transmission has been a perplexing and often controversial problem. In this issue of Neuron, Fernandez-Alfonso et al. present data indicating that at least two synaptic vesicle proteins, synaptotagmin 1 and VAMP-2, are present in a large pool on the synaptic and axonal plasma membrane and can interchange with recently exocytosed proteins. These findings suggest that a plasma membrane pool of synaptic vesicle proteins provides a reservoir that can facilitate rapid endocytosis.

Animals↗
PubMed2006

Expression profiling of a hypercontraction-induced myopathy in Drosophila suggests a compensatory cytoskeletal remodeling response.

Mutations that alter muscle contraction lead to a large array of diseases, including muscular dystrophies and cardiomyopathies. Although the molecular lesions underlying many hereditary muscle diseases are known, the downstream pathways that contribute to disease pathogenesis and compensatory muscle remodeling are poorly defined. We have recently identified and characterized mutations in Myosin Heavy Chain (Mhc) that lead to hypercontraction and subsequent degeneration of flight muscles in Drosophila. To characterize the genomic response to hypercontraction-induced myopathy, we performed expression analysis using Affymetrix high density oligonucleotide microarrays in Drosophila Mhc hypercontraction alleles. The altered transcriptional profile of dystrophic Mhc muscles suggests an actin-dependent remodeling of the muscle cytoskeleton. Specifically, a subset of the highly up-regulated transcripts is involved in actin regulation and structural support for the contractile machinery. In addition, we identified previously uncharacterized proteins with putative actin-interaction domains that are up-regulated in Mhc mutants and differentially expressed in muscles. Several of the up-regulated proteins, including the dystrophin-related protein, MSP-300, and the homolog of the neuronal activity-regulated protein, ARC, localize to specific subcellular muscle structures that may provide key structural sites for cytoskeletal remodeling in dystrophic muscles. Defining the genome-wide transcriptional response to muscle hypercontraction in Drosophila has revealed candidate loci that may participate in the pathogenesis of muscular dystrophy and in compensatory muscle repair pathways through modulation of the actin cytoskeleton.

Actins↗
PubMed2006

Development of a Drosophila seizure model for in vivo high-throughput drug screening.

An important application of model organisms in neurological research has been to identify and characterise therapeutic approaches for epilepsy, a recurrent seizure disorder that affects > 1% of the human population. Proconvulsant-treated rodent models have been widely used for antiepileptic drug discovery and development, but are not suitable for high-throughput screening. To generate a genetically tractable model that would be suitable for large-scale, high-throughput screening for antiepileptic drug candidates, we characterized a Drosophila chemical treatment model using the GABA(A) receptor antagonist picrotoxin. This proconvulsant, delivered to Drosophila larvae via simple feeding methods suitable for automated screening, generated robust generalised seizures with lethality occurring at doses between 0.3 and 0.5 mg/mL. Electrophysiological analysis of CNS motor neuron output in picrotoxin-treated larvae revealed generalised seizures within minutes of drug exposure. At subthreshold doses for seizure induction, picrotoxin produced an increased frequency of motor neuron action potential bursting, indicating that CNS GABAergic transmission regulates patterned activity. Mutants in the Drosophila Rdl GABA(A) receptor are resistant to picrotoxin, confirming that seizure induction occurs via a conserved GABA(A) receptor pathway. To validate the usefulness of this model for in vivo drug screening, we identified several classes of neuroactive antiepileptic compounds in a pilot screen, including phenytoin and nifedipine, which can rescue the seizures and lethal neurotoxicity induced by picrotoxin. The well-defined actions of picrotoxin in Drosophila and the ease with which compounds can be assayed for antiseizure activity makes this genetically tractable model attractive for high-throughput in vivo screens to identify novel anticonvulsants and seizure susceptibility loci.

Amino Acid Sequence↗
PubMed2005

Retrograde signaling by Syt 4 induces presynaptic release and synapse-specific growth.

The molecular pathways involved in retrograde signal transduction at synapses and the function of retrograde communication are poorly understood. Here, we demonstrate that postsynaptic calcium 2+ ion (Ca2+) influx through glutamate receptors and subsequent postsynaptic vesicle fusion trigger a robust induction of presynaptic miniature release after high-frequency stimulation at Drosophila neuromuscular junctions. An isoform of the synaptotagmin family, synaptotagmin 4 (Syt 4), serves as a postsynaptic Ca2+ sensor to release retrograde signals that stimulate enhanced presynaptic function through activation of the cyclic adenosine monophosphate (cAMP)-cAMP-dependent protein kinase pathway. Postsynaptic Ca2+ influx also stimulates local synaptic differentiation and growth through Syt 4-mediated retrograde signals in a synapse-specific manner.

Animals↗
PubMed2005

Genome-wide transcriptional changes associated with enhanced activity in the Drosophila nervous system.

Neuronal plasticity is an important feature of the developing brain and requires neuronal circuits to reconfigure their functional connectivity depending upon activity patterns. To explore changes in neuronal function that occur downstream of altered activity, we performed an expression analysis in Drosophila mutants with acute or chronic alterations in neuronal activity. We find that seizure induction leads to an overproliferation of synaptic connections, indicating that activity-dependent neuronal rewiring occurs in Drosophila. To analyze transcriptional recoding during altered neuronal activity, we performed genome-wide DNA microarray analysis following multiple seizure induction and recovery paradigms. Approximately 250 genes implicated in cell adhesion, membrane excitability, and cellular signaling are differentially regulated, including the Kek 2 neuronal cell adhesion protein, which, as we demonstrate, functions as a regulator of synaptic growth. These data identify a collection of activity-regulated transcripts that may link changes in neuronal firing patterns to transcription-dependent modulation of brain function, including activity-dependent synaptic rewiring.

Action Potentials↗
PubMed2004

Synaptotagmins are trafficked to distinct subcellular domains including the postsynaptic compartment.

The synaptotagmin family has been implicated in calcium-dependent neurotransmitter release, although Synaptotagmin 1 is the only isoform demonstrated to control synaptic vesicle fusion. Here, we report the characterization of the six remaining synaptotagmin isoforms encoded in the Drosophila genome, including homologues of mammalian Synaptotagmins 4, 7, 12, and 14. Like Synaptotagmin 1, Synaptotagmin 4 is ubiquitously present at synapses, but localizes to the postsynaptic compartment. The remaining isoforms were not found at synapses (Synaptotagmin 7), expressed at very low levels (Synaptotagmins 12 and 14), or in subsets of putative neurosecretory cells (Synaptotagmins alpha and beta). Consistent with their distinct localizations, overexpression of Synaptotagmin 4 or 7 cannot functionally substitute for the loss of Synaptotagmin 1 in synaptic transmission. Our results indicate that synaptotagmins are differentially distributed to unique subcellular compartments. In addition, the identification of a postsynaptic synaptotagmin suggests calcium-dependent membrane-trafficking functions on both sides of the synapse.

Animals↗
PubMed2004

A Drosophila temperature-sensitive seizure mutant in phosphoglycerate kinase disrupts ATP generation and alters synaptic function.

A novel paralytic mutant, nubian, was identified in a behavioral screen for conditional temperature-sensitive seizure mutants in Drosophila melanogaster. nubian mutants display reduced lifespan, abnormal motor behavior, altered synaptic structure, and defective neurotransmitter release. The nubian mutant disrupts phosphoglycerate kinase (PGK), an enzyme required for ATP generation in the terminal stage of the glycolytic pathway. Consistent with altered ATP generation in nubian animals, brain extracts show a threefold reduction in resting ATP levels compared with controls. Microarray analysis of nubian mutants reveals altered transcription of genes implicated in glucose and lipid metabolism. Disruption of ATP generation in nubian animals is accompanied by temperature-dependent defects in neuronal activity, with initial seizure activity, followed by an activity-dependent loss of synaptic transmission. nubian mutants also display structural defects at the synapse, with larger varicosity size but normal varicosity number, indicating that these synaptic parameters are regulated independently. Both exocytotic (NSF) and endocytotic (dynamin) ATPase/GTPase activity are required for normal synaptic transmission. Biochemical and physiological analyses indicate that synaptic defects in nubian animals are secondary to defective endocytosis, suggesting that endocytotic pathways may be generally more sensitive to altered ATP levels than those used for exocytosis. Alterations in ATP metabolism likely disrupt similar pathways in humans, because PGK deficiency is associated with mental retardation, seizures, and exercise intolerance. Given the behavioral similarities between disruptions of PGK function in Drosophila and humans, the analysis of nubian animals may reveal conserved neuronal responses associated with altered ATP generation within the brain.

Adenosine Triphosphate↗
PubMed2004

Characterization of a hypercontraction-induced myopathy in Drosophila caused by mutations in Mhc.

The Myosin heavy chain (Mhc) locus encodes the muscle-specific motor mediating contraction in Drosophila. In a screen for temperature-sensitive behavioral mutants, we have identified two dominant Mhc alleles that lead to a hypercontraction-induced myopathy. These mutants are caused by single point mutations in the ATP binding/hydrolysis domain of Mhc and lead to degeneration of the flight muscles. Electrophysiological analysis in the adult giant fiber flight circuit demonstrates temperature-dependent seizure activity that requires neuronal input, as genetic blockage of neuronal activity suppresses the electrophysiological seizure defects. Intracellular recordings at the third instar neuromuscular junction show spontaneous muscle movements in the absence of neuronal stimulation and extracellular Ca2+, suggesting a dysregulation of intracellular calcium homeostasis within the muscle or an alteration of the Ca2+ dependence of contraction. Characterization of these new Mhc alleles suggests that hypercontraction occurs via a mechanism, which is molecularly distinct from mutants identified previously in troponin I and troponin T.

Adenosine Triphosphate↗
PubMed2004

Cytoplasmic aggregates trap polyglutamine-containing proteins and block axonal transport in a Drosophila model of Huntington's disease.

Huntington's disease is an autosomal dominant neurodegenerative disorder caused by expansion of a polyglutamine tract in the huntingtin protein that results in intracellular aggregate formation and neurodegeneration. Pathways leading from polyglutamine tract expansion to disease pathogenesis remain obscure. To elucidate how polyglutamine expansion causes neuronal dysfunction, we generated Drosophila transgenic strains expressing human huntingtin cDNAs encoding pathogenic (Htt-Q128) or nonpathogenic proteins (Htt-Q0). Whereas expression of Htt-Q0 has no discernible effect on behavior, lifespan, or neuronal morphology, pan-neuronal expression of Htt-Q128 leads to progressive loss of motor coordination, decreased lifespan, and time-dependent formation of huntingtin aggregates specifically in the cytoplasm and neurites. Huntingtin aggregates sequester other expanded polyglutamine proteins in the cytoplasm and lead to disruption of axonal transport and accumulation of aggregates at synapses. In contrast, Drosophila expressing an expanded polyglutamine tract alone, or an expanded polyglutamine tract in the context of the spinocerebellar ataxia type 3 protein, display only nuclear aggregates and do not disrupt axonal trafficking. Our findings indicate that nonnuclear events induced by cytoplasmic huntingtin aggregation play a central role in the progressive neurodegeneration observed in Huntington's disease.

Animals↗
PubMed2004

Nervous wreck, an SH3 adaptor protein that interacts with Wsp, regulates synaptic growth in Drosophila.

We describe the isolation and characterization of nwk (nervous wreck), a temperature-sensitive paralytic mutant that causes excessive growth of larval neuromuscular junctions (NMJs), resulting in increased synaptic bouton number and branch formation. Ultrastructurally, mutant boutons have reduced size and fewer active zones, associated with a reduction in synaptic transmission. nwk encodes an FCH and SH3 domain-containing adaptor protein that localizes to the periactive zone of presynaptic terminals and binds to the Drosophila ortholog of Wasp (Wsp), a key regulator of actin polymerization. wsp null mutants display synaptic overgrowth similar to nwk and enhance the nwk morphological phenotype in a dose-dependent manner. Evolutionarily, Nwk belongs to a previously undescribed family of adaptor proteins that includes the human srGAPs, which regulate Rho activity downstream of Robo receptors. We propose that Nwk controls synapse morphology by regulating actin dynamics downstream of growth signals in presynaptic terminals.

Adaptor Proteins, Vesicular Transport↗
PubMed2003

Presynaptic N-type calcium channels regulate synaptic growth.

Voltage-gated calcium channels couple changes in membrane potential to neuronal functions regulated by calcium, including neurotransmitter release. Here we report that presynaptic N-type calcium channels not only control neurotransmitter release but also regulate synaptic growth at Drosophila neuromuscular junctions. In a screen for behavioral mutants that disrupt synaptic transmission, an allele of the N-type calcium channel locus (Dmca1A) was identified that caused synaptic undergrowth. The underlying molecular defect was identified as a neutralization of a charged residue in the third S4 voltage sensor. RNA interference reduction of N-type calcium channel expression also reduced synaptic growth. Hypomorphic mutations in syntaxin-1A or n-synaptobrevin, which also disrupt neurotransmitter release, did not affect synapse proliferation at the neuromuscular junction, suggesting calcium entry through presynaptic N-type calcium channels, not neurotransmitter release per se, is important for synaptic growth. The reduced synapse proliferation in Dmca1A mutants is not due to increased synapse retraction but instead reflects a role for calcium influx in synaptic growth mechanisms. These results suggest N-type channels participate in synaptic growth through signaling pathways that are distinct from those that mediate neurotransmitter release. Linking presynaptic voltage-gated calcium entry to downstream calcium-sensitive synaptic growth regulators provides an efficient activity-dependent mechanism for modifying synaptic strength.

Alleles↗
PubMed2003

Is synaptotagmin the calcium sensor?

After much debate, recent progress indicates that the synaptic vesicle protein synaptotagmin I probably functions as the calcium sensor for synchronous neurotransmitter release. Following calcium influx into presynaptic terminals, synaptotagmin I rapidly triggers the fusion of synaptic vesicles with the plasma membrane and underlies the fourth-order calcium cooperativity of release. Biochemical and genetic studies suggest that lipid and SNARE interactions underlie synaptotagmin's ability to mediate the incredible speed of vesicle fusion that is the hallmark of fast synaptic transmission.

Animals↗
PubMed2002

Synaptotagmin I functions as a calcium sensor to synchronize neurotransmitter release.

To characterize Ca(2+)-mediated synaptic vesicle fusion, we analyzed Drosophila synaptotagmin I mutants deficient in specific interactions mediated by its two Ca(2+) binding C2 domains. In the absence of synaptotagmin I, synchronous release is abolished and a kinetically distinct delayed asynchronous release pathway is uncovered. Synapses containing only the C2A domain of synaptotagmin partially recover synchronous fusion, but have an abolished Ca(2+) cooperativity. Mutants that disrupt Ca(2+) sensing by the C2B domain have synchronous release with normal Ca(2+) cooperativity, but with reduced release probability. Our data suggest the Ca(2+) cooperativity of neurotransmitter release is likely mediated through synaptotagmin-SNARE interactions, while phospholipid binding and oligomerization trigger rapid fusion with increased release probability. These results indicate that synaptotagmin is the major Ca(2+) sensor for evoked release and functions to trigger synchronous fusion in response to Ca(2+), while suppressing asynchronous release.

Animals↗