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1.
eNeuro ; 3(2)2016.
Article in English | MEDLINE | ID: mdl-27022630

ABSTRACT

Synaptic signaling involves the release of neurotransmitter from presynaptic active zones (AZs). Proteins that regulate vesicle exocytosis cluster at AZs, composing the cytomatrix at the active zone (CAZ). At the Drosophila neuromuscular junction (NMJ), the small GTPase Rab3 controls the distribution of CAZ proteins across release sites, thereby regulating the efficacy of individual AZs. Here we identify Rab3-GEF as a second protein that acts in conjunction with Rab3 to control AZ protein composition. At rab3-GEF mutant NMJs, Bruchpilot (Brp) and Ca(2+) channels are enriched at a subset of AZs, leaving the remaining sites devoid of key CAZ components in a manner that is indistinguishable from rab3 mutant NMJs. As the Drosophila homologue of mammalian DENN/MADD and Caenorhabditis elegans AEX-3, Rab3-GEF is a guanine nucleotide exchange factor (GEF) for Rab3 that stimulates GDP to GTP exchange. Mechanistic studies reveal that although Rab3 and Rab3-GEF act within the same mechanism to control AZ development, Rab3-GEF is involved in multiple roles. We show that Rab3-GEF is required for transport of Rab3. However, the synaptic phenotype in the rab3-GEF mutant cannot be fully explained by defective transport and loss of GEF activity. A transgenically expressed GTP-locked variant of Rab3 accumulates at the NMJ at wild-type levels and fully rescues the rab3 mutant but is unable to rescue the rab3-GEF mutant. Our results suggest that although Rab3-GEF acts upstream of Rab3 to control Rab3 localization and likely GTP-binding, it also acts downstream to regulate CAZ development, potentially as a Rab3 effector at the synapse.


Subject(s)
Gene Expression Regulation, Developmental/genetics , Mutation/genetics , Neuromuscular Junction/cytology , Presynaptic Terminals/physiology , rab3 GTP-Binding Proteins/metabolism , Action Potentials/genetics , Analysis of Variance , Animals , Animals, Genetically Modified , Drosophila , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Microscopy, Confocal , Neuromuscular Junction/genetics , Neurons/physiology , Patch-Clamp Techniques , rab3 GTP-Binding Proteins/genetics
2.
PLoS One ; 10(8): e0136938, 2015.
Article in English | MEDLINE | ID: mdl-26317909

ABSTRACT

At synapses, the release of neurotransmitter is regulated by molecular machinery that aggregates at specialized presynaptic release sites termed active zones. The complement of active zone proteins at each site is a determinant of release efficacy and can be remodeled to alter synapse function. The small GTPase Rab3 was previously identified as playing a novel role that controls the distribution of active zone proteins to individual release sites at the Drosophila neuromuscular junction. Rab3 has been extensively studied for its role in the synaptic vesicle cycle; however, the mechanism by which Rab3 controls active zone development remains unknown. To explore this mechanism, we conducted a mutational analysis to determine the molecular and structural requirements of Rab3 function at Drosophila synapses. We find that GTP-binding is required for Rab3 to traffick to synapses and distribute active zone components across release sites. Conversely, the hydrolytic activity of Rab3 is unnecessary for this function. Through a structure-function analysis we identify specific residues within the effector-binding switch regions that are required for Rab3 function and determine that membrane attachment is essential. Our findings suggest that Rab3 controls the distribution of active zone components via a vesicle docking mechanism that is consistent with standard Rab protein function.


Subject(s)
DNA Mutational Analysis/methods , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Neuromuscular Junction/metabolism , rab3 GTP-Binding Proteins/genetics , rab3 GTP-Binding Proteins/metabolism , Animals , Binding Sites , Drosophila Proteins/chemistry , Drosophila melanogaster/genetics , Neuromuscular Junction/physiology , Protein Binding , Synaptic Vesicles/metabolism , rab3 GTP-Binding Proteins/chemistry
3.
J Neurosci ; 32(47): 16586-96, 2012 Nov 21.
Article in English | MEDLINE | ID: mdl-23175814

ABSTRACT

Synaptic communication requires the controlled release of synaptic vesicles from presynaptic axon terminals. Release efficacy is regulated by the many proteins that comprise the presynaptic release apparatus, including Ca(2+) channels and proteins that influence Ca(2+) channel accumulation at release sites. Here we identify Drosophila RIM (Rab3 interacting molecule) and demonstrate that it localizes to active zones at the larval neuromuscular junction. In Drosophila RIM mutants, there is a large decrease in evoked synaptic transmission because of a significant reduction in both the clustering of Ca(2+) channels and the size of the readily releasable pool of synaptic vesicles at active zones. Hence, RIM plays an evolutionarily conserved role in regulating synaptic calcium channel localization and readily releasable pool size. Because RIM has traditionally been studied as an effector of Rab3 function, we investigate whether RIM is involved in the newly identified function of Rab3 in the distribution of presynaptic release machinery components across release sites. Bruchpilot (Brp), an essential component of the active zone cytomatrix T bar, is unaffected by RIM disruption, indicating that Brp localization and distribution across active zones does not require wild-type RIM. In addition, larvae containing mutations in both RIM and rab3 have reduced Ca(2+) channel levels and a Brp distribution that is very similar to that of the rab3 single mutant, indicating that RIM functions to regulate Ca(2+) channel accumulation but is not a Rab3 effector for release machinery distribution across release sites.


Subject(s)
Calcium Channels/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila/physiology , Neuromuscular Junction/metabolism , rab3 GTP-Binding Proteins/genetics , rab3 GTP-Binding Proteins/metabolism , Animals , Cloning, Molecular , DNA Mutational Analysis , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Drosophila Proteins/physiology , Image Processing, Computer-Assisted , Immunohistochemistry , Larva , Microscopy, Confocal , Microscopy, Electron , Patch-Clamp Techniques , Synaptic Vesicles/metabolism , Synaptic Vesicles/ultrastructure , rab3 GTP-Binding Proteins/physiology
4.
J Neurosci ; 31(42): 15026-34, 2011 Oct 19.
Article in English | MEDLINE | ID: mdl-22016536

ABSTRACT

Synaptic connections can be stably maintained for prolonged periods, yet can be rapidly disassembled during the developmental refinement of neural circuitry and following cytological insults that lead to neurodegeneration. To date, the molecular mechanisms that determine whether a synapse will persist versus being remodeled or eliminated remain poorly understood. Mutations in Drosophila stathmin were isolated in two independent genetic screens that sought mutations leading to impaired synapse stability at the Drosophila neuromuscular junction (NMJ). Here we demonstrate that Stathmin, a protein that associates with microtubules and can function as a point of signaling integration, is necessary to maintain the stability of the Drosophila NMJ. We show that Stathmin protein is widely distributed within motoneurons and that loss of Stathmin causes impaired NMJ growth and stability. In addition, we show that stathmin mutants display evidence of defective axonal transport, a common feature associated with neuronal degeneration and altered synapse stability. The disassembly of the NMJ in stathmin includes a predictable sequence of cytological events, suggesting that a common program of synapse disassembly is induced following the loss of Stathmin protein. These data define a required function for Stathmin during synapse maintenance in a model system in which there is only a single stathmin gene, enabling future genetic investigation of Stathmin function with potential relevance to the cause and progression of neuromuscular degenerative disease.


Subject(s)
Neuromuscular Junction/physiology , Stathmin/metabolism , Analysis of Variance , Animals , Animals, Genetically Modified , Axons/physiology , Drosophila , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Microscopy, Confocal , Mutation/genetics , Neuromuscular Junction/cytology , Neuromuscular Junction/genetics , Presynaptic Terminals/metabolism , RNA Interference/physiology , Stathmin/genetics , Vesicular Glutamate Transport Proteins/genetics
5.
Neuron ; 64(5): 663-77, 2009 Dec 10.
Article in English | MEDLINE | ID: mdl-20005823

ABSTRACT

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.


Subject(s)
Gene Expression Regulation/physiology , Neuromuscular Junction/cytology , Nonlinear Dynamics , Presynaptic Terminals/metabolism , rab3 GTP-Binding Proteins/metabolism , Animals , Animals, Genetically Modified , Calcium/pharmacology , Drosophila , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/genetics , Gene Expression Regulation/genetics , Green Fluorescent Proteins/genetics , Microscopy, Electron, Transmission/methods , Motor Endplate/metabolism , Motor Endplate/ultrastructure , Mutation/genetics , Neuromuscular Junction/drug effects , Presynaptic Terminals/ultrastructure , Receptors, Glutamate/genetics , rab3 GTP-Binding Proteins/genetics
6.
J Neurosci ; 26(16): 4256-65, 2006 Apr 19.
Article in English | MEDLINE | ID: mdl-16624946

ABSTRACT

Recent findings suggest that the neurexin-neuroligin link promotes both GABAergic and glutamatergic synaptogenesis, but the mechanism by which neurexins influence the clustering of appropriate neuroligins and postsynaptic differentiation remains unclear. Previous studies suggested that the presence or absence of alternatively spliced residues at splice site 4 (S4) in the neurexin LNS domain may regulate neurexin function. We demonstrate that addition of the S4 insert selectively reduces the ability of neurexin-1beta to cluster neuroligin-1/3/4 and glutamatergic postsynaptic proteins, although clustering of neuroligin-2 and GABAergic postsynaptic proteins remain strong. Furthermore, addition of the S4 insert decreases the binding affinity of neurexin-1beta to neuroligins-1 and -4 but has little effect on binding to neuroligins-2 and -3. Additional structure-function studies reveal the neurexin binding interface mediating synaptogenic activity to be composed primarily of residues in the beta2beta3, beta6beta7, and beta10beta11 loops on one rim of the LNS domain beta sandwich. Mutation of two predicted Ca(2+)-binding residues disrupts postsynaptic protein clustering and binding to neuroligins, consistent with previous findings that neurexin-neuroligin binding is Ca2+ dependent. Glutamatergic postsynaptic clustering was more readily disrupted by the mutagenesis than GABAergic postsynaptic protein clustering. Perhaps neurexins-neuroligins, or neurexin-1beta at least, is most important for GABA synapse formation or controlling the balance of GABA and glutamate synapses. These results suggest that differential neurexin-neuroligin binding affinities and splice variations may play an instructive role in postsynaptic differentiation.


Subject(s)
DNA, Recombinant/genetics , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , RNA Splice Sites/genetics , Synapses/genetics , Amino Acid Sequence/physiology , Animals , COS Cells , Cells, Cultured , Chlorocebus aethiops , DNA, Recombinant/metabolism , Glutamic Acid/physiology , Molecular Sequence Data , Nerve Tissue Proteins/physiology , Protein Structure, Tertiary/genetics , Rats , Structure-Activity Relationship , Synapses/drug effects , Synapses/physiology , gamma-Aminobutyric Acid/pharmacology
7.
Trends Neurosci ; 29(1): 8-20, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16337695

ABSTRACT

Central neurons develop and maintain molecularly distinct synaptic specializations for excitatory and inhibitory transmitters, often only microns apart on their dendritic arbor. Progress towards understanding the molecular basis of synaptogenesis has come from several recent studies using a coculture system of non-neuronal cells expressing molecules that generate presynaptic or postsynaptic "hemi-synapses" on contacting neurons. Together with molecular properties of these protein families, such studies have yielded interesting clues to how glutamatergic and GABAergic synapses are assembled. Other clues come from heterochronic cultures, manipulations of activity in subsets of neurons in a network, and of course many in vivo studies. Taking into account these data, we consider here how basic parameters of synapses--competence, placement, composition, size and longevity--might be determined.


Subject(s)
Glutamates/metabolism , Models, Neurological , Neurons/physiology , Neurotransmitter Agents/metabolism , Synapses/physiology , Synaptic Transmission/physiology , gamma-Aminobutyric Acid/metabolism , Animals , Cells, Cultured , Humans
8.
Cell ; 119(7): 1013-26, 2004 Dec 29.
Article in English | MEDLINE | ID: mdl-15620359

ABSTRACT

Formation of synaptic connections requires alignment of neurotransmitter receptors on postsynaptic dendrites opposite matching transmitter release sites on presynaptic axons. beta-neurexins and neuroligins form a trans-synaptic link at glutamate synapses. We show here that neurexin alone is sufficient to induce glutamate postsynaptic differentiation in contacting dendrites. Surprisingly, neurexin also induces GABA postsynaptic differentiation. Conversely, neuroligins induce presynaptic differentiation in both glutamate and GABA axons. Whereas neuroligins-1, -3, and -4 localize to glutamate postsynaptic sites, neuroligin-2 localizes primarily to GABA synapses. Direct aggregation of neuroligins reveals a linkage of neuroligin-2 to GABA and glutamate postsynaptic proteins, but the other neuroligins only to glutamate postsynaptic proteins. Furthermore, mislocalized expression of neuroligin-2 disperses postsynaptic proteins and disrupts synaptic transmission. Our findings indicate that the neurexin-neuroligin link is a core component mediating both GABAergic and glutamatergic synaptogenesis, and differences in isoform localization and binding affinities may contribute to appropriate differentiation and specificity.


Subject(s)
Cell Differentiation/drug effects , Glutamic Acid/metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/pharmacology , Synapses/drug effects , gamma-Aminobutyric Acid/metabolism , Amino Acid Motifs/genetics , Animals , COS Cells , Cell Adhesion Molecules, Neuronal , Coculture Techniques , Dendrites/metabolism , Dystroglycans/metabolism , Hippocampus/cytology , Hippocampus/drug effects , Hippocampus/metabolism , Humans , Mice , Mutation , Nerve Tissue Proteins/genetics , Protein Binding , Rats , Synapses/metabolism
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