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Presynaptic Spectrin Is Essential for Synapse Stabilization

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Presynaptic Spectrin Is Essential for Synapse Stabilization View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Current Biology, Vol. 15, 918–928, May 24, 2005, ©2005 Elsevier Ltd All rights reserved. DOI 10.1016/j.cub.2005.04.030 Presynaptic Spectrin Is Essential for Synapse Stabilization Jan Pielage,1 Richard D. Fetter,2 aptic nerve terminal. The basic unit of the spectrin skel- and Graeme W. Davis1,* eton is a heterotetramer composed of α- and β-Spectrin 1Department of Biochemistry and Biophysics subunits. These heterotetramers can interact with short Program in Neuroscience actin filaments to form a spectrin-actin filamentous net- University of California, San Francisco work that is localized to the plasma membrane [5]. San Francisco, California 94143 Spectrin can directly associate with membrane phos- 2 Laboratory of Neural Circuits and Behavior pholipids and may provide support for cell shape in this The Rockefeller University manner [5]. The spectrin skeleton has also been termed 1230 York Avenue a “protein accumulation machine” because of its in- New York, New York 10021 volvement in ion-channel, cell-adhesion-molecule, and adaptor-protein localization [6]. In the nervous system, the spectrin skeleton is abun- Summary dant both pre- and postsynaptically at both central and peripheral synapses [5, 7, 8]. However, little is known Background: Precise neural circuitry is established and regarding the function of spectrin in the postembryonic maintained through a regulated balance of synapse nervous system owing to the embryonic lethality of mu- stabilization and disassembly. Currently, little is known tations in most spectrin isoforms. In Drosophila, null about the molecular mechanisms that specify synapse mutations in a- or b-spectrin are late embryonic/early stability versus disassembly. first instar lethal [9, 10]. As a result, prior work has been Results: Here, we demonstrate that presynaptic spectrin restricted to the newly formed NMJ, where spectrin is is an essential scaffold that is required to maintain syn- present both pre- and postsynaptically [11]. Surpris- apse stability at the Drosophila neuromuscular junction ingly, no change in synapse morphology, synapse ultra- (NMJ). Loss of presynaptic spectrin leads to synapse structure, or receptor clustering was observed [11]. disassembly and ultimately to the elimination of the Similar conclusions were made in C. elegans [12]. NMJ. Synapse elimination is documented through light- These prior studies characterized the early develop- level, ultrastructural, and electrophysiological assays. mental requirements of the spectrin skeleton during These combined assays reveal that impaired neuro- synapse formation, but they were unable to assess the transmission is secondary to synapse retraction. We function of spectrin at mature synapses in the postem- demonstrate that loss of presynaptic, but not postsyn- bryonic nervous system. Here, we document the use of aptic, spectrin leads to the disorganization and elimina- transgenic dsRNA reagents that allow us to eliminate tion of essential synaptic cell-adhesion molecules. In spectrin protein selectively during postembryonic de- addition, we provide evidence of altered axonal trans- velopment from either the pre- or postsynaptic side of port and disrupted synaptic microtubules as events the synapse. We show that the presynaptic spectrin that contribute to synapse retraction in animals lacking skeleton is essential during postembryonic develop- presynaptic spectrin. ment for the maintenance of the Drosophila NMJ. Conclusions: Our data suggest that presynaptic spec- trin functions as an essential presynaptic scaffold that Results may link synaptic cell adhesion with the stabilization of the underlying microtubule cytoskeleton. We have generated transgenic animals that allow tis- sue-specific expression of either a- or b-spectrin dsRNA Introduction with the GAL4/UAS expression system (see Experimen- tal Procedures). It has been clearly demonstrated that Throughout the nervous system, the development of transgenically expressed dsRNA can function cell au- neural circuitry involves exuberant synaptogenesis fol- tonomously in Drosophila and is highly sequence spe- lowed by the selective elimination of a subset of these cific [13–15]. We expressed either UAS-a-spectrin previously functional synapses [1–4]. Currently, we dsRNA or UAS-b-spectrin dsRNA in the embryo with know very little about the mechanisms that stabilize cell-type-specific GAL4 drivers and observed rapid, synaptic connections and equally little regarding the specific, and cell-autonomous knockdown of spectrin mechanisms that disassemble previously stable, func- mRNA and protein (data not shown). We observe effi- tional synaptic connections [2]. One way to approach cient knockdown of mRNA and protein in w6hrin this problem is to identify genes that when mutated or these cells. We next tested whether our ability to elimi- knocked down cause the inappropriate disassembly of nate spectrin protein with dsRNA can recapitulate the a previously stable synapse. This approach will identify late-embryonic/early-larval lethality observed in a- and genes necessary for synapse stabilization and may b-spectrin null mutations [9, 10]. Ubiquitous expression identify candidate genes involved in neurodegenera- of a- or b-spectrin dsRNA leads to lethality at early tive disease. stages of larval development. Thus, we nearly recapitu- Here, we demonstrate that the presynaptic spectrin late the null mutant lethal phenotype, and the slight dif- skeleton is essential for the stabilization of the presyn- ference in lethal phase can likely be attributed to the delay in dsRNA expression in comparison to the effects *Correspondence: [email protected] of a null mutation that blocks zygotic gene expression. Spectrin Stabilizes the Synapse 919 Figure 1. Selective Elimination of α- and β-Spectrin at the NMJ (A–C) A wild-type synapse at muscle 4 is stained with anti-β-Spectrin ([A], green) and anti-HRP ([B], red). The merged image is shown in (C). β-Spectrin staining is detected in the presynaptic motoneuron axon ([A] and [B], arrows) and in muscle. (D–F) Expression of UAS-b-spectrin dsRNA in the presynaptic neuron (sca-GAL4) results in the elimination of presynaptic β-Spectrin protein. Staining is no longer observed in the presynaptic motoneuron axon ([D], arrowhead) that can be identified by anti-HRP staining ([E], ar- rowhead). (G–I) Expression of UAS-b-spectrin-dsRNA in the postsynaptic muscle (BG57-GAL4) results in the elimination of β-Spectrin protein from the postsynaptic muscle ([G] and [H], asterisks), including sites where the presynaptic nerve terminal remains ([H], asterisk). (J and K) Synaptic stainings are shown at higher exposures. Presynaptic α-Spectrin staining can be observed after elimination of postsynaptic α- Spectrin. Presynaptic α-Spectrin staining is continuous with axonal staining prior to entry into the muscle field ([J] and [K], arrowheads). α-Spectrin staining is also contained within the presynaptic-membrane boundary defined by anti-HRP ([J] and [K], arrow). Neuronal elimination of either α-orβ-Spectrin causes to visualize presynaptic spectrin. In Figures 1A–1I, lethality at late larval stages, with a small portion of ani- staining intensities are optimized to visualize protein mals surviving to early adult stages. Thus, both a- and levels at the wild-type NMJ that has high levels of b-spectrin are essential neuronal genes. spectrin expression. At longer exposure lengths, pre- synaptic spectrin immunoreactivity can be clearly ob- Analysis of the Postembryonic Synaptic served in the absence of postsynaptic spectrin protein Spectrin Skeleton (Figures 1J and 1K). Although we cannot formally rule Three spectrin genes are present in the Drosophila ge- out that some of this staining is postsynaptic, several nome [9, 10, 16]. At the larval NMJ, both α- and β-Spec- lines of evidence suggest that this staining is primarily trin are highly abundant and colocalize (Figure 1; see presynaptic. First, in these examples, α-Spectrin stain- also [11]) whereas βH-Spectrin is absent (data not ing at the nerve terminal does not extend beyond the shown). Postsynaptic α- and β-Spectrin can be ob- boundary of the presynaptic-membrane marker anti- served throughout the muscle and are concentrated to HRP (Figures 1J and 1K, arrows). In addition, the ob- the postsynaptic membranes surrounding the NMJ served α-Spectrin staining is continuous with staining (Figure 1). Presynaptic α- and β-Spectrin can be ob- that originates within the presynaptic axon prior to en- served in the motoneuron axon (Figure 1), and we pre- try into the muscle (Figures 1J and 1K, arrowheads). sent evidence below that this staining continues into the presynaptic boutons (Figures 1J and 1K). Loss of Presynaptic Spectrin Leads to Synapse Using the UAS-spectrin dsRNA approach, we can Disassembly and Elimination eliminate either α-Spectrin or β-Spectrin protein specif- Loss of presynaptic spectrin (either α-orβ-Spectrin) ically from either the pre- or postsynaptic side of the leads to presynaptic retraction and synapse elimination neuromuscular junction (Figures 1D–1I). Our ability to at the NMJ. We have previously published an assay for selectively eliminate postsynaptic spectrin enables us synapse disassembly at the Drosophila NMJ [17]. This Current Biology 920 assay is based on the observation that the gradual as- trin dsRNA presynaptically. We have compared the sembly of the postsynaptic muscle membrane
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