Yogesh P. Wairkar

Highwire Restrains Synaptic Growth by Attenuating a MAP Kinase Signal

Highwire is an extremely large, evolutionarily conserved E3 ubiquitin ligase that negatively regulates synaptic growth at the Drosophila NMJ. Highwire has been proposed to restrain synaptic growth by downregulating a synaptogenic signal. Here we identify such a downstream signaling pathway. A screen for suppressors of the highwire synaptic overgrowth phenotype yielded mutations in wallenda, a MAP kinase kinase kinase (MAPKKK) homologous to vertebrate DLK and LZK. wallenda is both necessary for highwire synaptic overgrowth and sufficient to promote synaptic overgrowth, and synaptic levels of Wallenda protein are controlled by Highwire and ubiquitin hydrolases. highwire synaptic overgrowth requires the MAP kinase JNK and the transcription factor Fos. These results suggest that Highwire controls structural plasticity of the synapse by regulating gene expression through a MAP kinase signaling pathway. In addition to controlling synaptic growth, Highwire promotes synaptic function through a separate pathway that does not require wallenda.

Eps15 and Dap160 control synaptic vesicle membrane retrieval and synapse development

Epidermal growth factor receptor pathway substrate clone 15 (Eps15) is a protein implicated in endocytosis, endosomal protein sorting, and cytoskeletal organization. Its role is, however, still unclear, because of reasons including limitations of dominant-negative experiments and apparent redundancy with other endocytic proteins. We generated Drosophila eps15-null mutants and show that Eps15 is required for proper synaptic bouton development and normal levels of synaptic vesicle (SV) endocytosis. Consistent with a role in SV endocytosis, Eps15 moves from the center of synaptic boutons to the periphery in response to synaptic activity. The endocytic protein, Dap160/intersectin, is a major binding partner of Eps15, and eps15 mutants phenotypically resemble dap160 mutants. Analyses of eps15 dap160 double mutants suggest that Eps15 functions in concert with Dap160 during SV endocytosis. Based on these data, we hypothesize that Eps15 and Dap160 promote the efficiency of endocytosis from the plasma membrane by maintaining high concentrations of multiple endocytic proteins, including dynamin, at synapses.

Highwire Function at the Drosophila Neuromuscular Junction: Spatial, Structural, and Temporal Requirements

Highwire is a huge, evolutionarily conserved protein that is required to restrain synaptic growth and promote synaptic transmission at the Drosophila neuromuscular junction. Current models of highwire function suggest that it may act as a ubiquitin ligase to regulate synaptic development. However, it is not known in which cells highwire functions, whether its putative ligase domain is required for function, or whether highwire regulates the synapse during development or alternatively sets cell fate in the embryo. We performed a series of transgenic rescue experiments to test the spatial, structural, and temporal requirements for highwire function. We find that presynaptic activity of highwire is both necessary and sufficient to regulate both synapse morphology and physiology. The Highwire RING domain, which is postulated to function as an E3 ubiquitin ligase, is required for highwire function. In addition, highwire acts throughout larval development to regulate synaptic morphology and function. Finally, we show that the morphological and physiological phenotypes of highwire mutants have different dosage and temporal requirements for highwire, demonstrating that highwire may independently regulate the molecular pathways controlling synaptic growth and function.

Drosophila Vps35 function is necessary for normal endocytic trafficking and actin cytoskeleton organisation

To identify novel proteins required for receptor-mediated endocytosis, we have developed an RNAi-based screening method in Drosophila S2 cells, based on uptake of a scavenger receptor ligand. Some known endocytic proteins are essential for endocytosis in this assay, including clathrin and α-adaptin; however, other proteins important for synaptic vesicle endocytosis are not required. In a small screen for novel endocytic proteins, we identified the Drosophila homologue of Vps35, a component of the retromer complex, involved in endosome-to-Golgi trafficking. Loss of Vps35 inhibits scavenger receptor ligand endocytosis, and causes mislocalisation of a number of receptors and endocytic proteins. Vps35 has tumour suppressor properties because its loss leads to overproliferation of blood cells in larvae. Its loss also causes signalling defects at the neuromuscular junction, including upregulation of TGFβ/BMP signalling and excessive formation of synaptic terminals. Vps35 negatively regulates actin polymerisation, and genetic interactions suggest that some of the endocytic and signalling defects of vps35 mutants are due to this function.

Unc-51 Controls Active Zone Density and Protein Composition by Downregulating ERK Signaling

Efficient synaptic transmission requires the apposition of neurotransmitter release sites opposite clusters of postsynaptic neurotransmitter receptors. Transmitter is released at active zones, which are composed of a large complex of proteins necessary for synaptic development and function. Many active zone proteins have been identified, but little is known of the mechanisms that ensure that each active zone receives the proper complement of proteins. Here we use a genetic analysis in Drosophila to demonstrate that the serine threonine kinase Unc-51 acts in the presynaptic motoneuron to regulate the localization of the active zone protein Bruchpilot opposite to glutamate receptors at each synapse. In the absence of Unc-51, many glutamate receptor clusters are unapposed to Bruchpilot, and ultrastructural analysis demonstrates that fewer active zones contain dense body T-bars. In addition to the presence of these aberrant synapses, there is also a decrease in the density of all synapses. This decrease in synaptic density and abnormal active zone composition is associated with impaired evoked transmitter release. Mechanistically, Unc-51 inhibits the activity of the MAP kinase ERK to promote synaptic development. In the unc-51 mutant, increased ERK activity leads to the decrease in synaptic density and the absence of Bruchpilot from many synapses. Hence, activated ERK negatively regulates synapse formation, resulting in either the absence of active zones or the formation of active zones without their proper complement of proteins. The Unc-51-dependent inhibition of ERK activity provides a potential mechanism for synapse-specific control of active zone protein composition and release probability.

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.

Synaptic Defects in a Drosophila Model of Congenital Muscular Dystrophy

The congenital muscular dystrophies present in infancy with muscle weakness and are often associated with mental retardation. Many of these inherited disorders share a common etiology: defective O-glycosylation of α-dystroglycan, a component of the dystrophin complex. Protein-O-mannosyl transferase 1 (POMT1) is the first enzyme required for the glycosylation of α-dystroglycan, and mutations in the POMT1 gene can lead to both Walker-Warburg syndrome (WWS) and limb girdle muscular dystrophy type 2K (LGMD2K). WWS is associated with severe mental retardation and major structural abnormalities in the brain; however, LGMD2K patients display a more mild retardation with no obvious structural defects in the brain. In a screen for synaptic mutants in Drosophila, we identified mutations in the Drosophila ortholog of POMT1, dPOMT1. Because synaptic defects are a plausible cause of mental retardation, we investigated the molecular and physiological defects associated with loss of dPOMT1 in Drosophila. In dPOMT1 mutants, there is a decrease in the efficacy of synaptic transmission and a change in the subunit composition of the postsynaptic glutamate receptors at the neuromuscular junction. We demonstrate that dPOMT1 is required to glycosylate the Drosophila dystroglycan ortholog Dg in vivo, and that this is the likely cause of these synaptic defects because (1) mutations in Dg lead to similar synaptic defects and (2) genetic interaction studies suggest that dPOMT1 and Dg function in the same pathway. These results are consistent with the model that dPOMT1-dependent glycosylation of Dg is necessary for proper synaptic function and raise the possibility that similar synaptic defects occur in the congenital muscular dystrophies.

The B′ Protein Phosphatase 2A Regulatory Subunit well-rounded Regulates Synaptic Growth and Cytoskeletal Stability at the Drosophila Neuromuscular Junction

Synaptic growth is essential for the development and plasticity of neural circuits. To identify molecular mechanisms regulating synaptic growth, we performed a gain-of-function screen for synapse morphology mutants at the Drosophila neuromuscular junction (NMJ). We isolated a B′ regulatory subunit of protein phosphatase 2A (PP2A) that we have named well-rounded (wrd). Neuronal overexpression of wrd leads to overgrowth of the synaptic terminal. Endogenous Wrd protein is present in the larval nervous system and muscle and is enriched at central and neuromuscular synapses. wrd is required for normal synaptic development; in its absence, there are fewer synaptic boutons and there is a decrease in synaptic strength. wrd functions presynaptically to promote normal synaptic growth and postsynaptically to maintain normal levels of evoked transmitter release. In the absence of wrd, the presynaptic cytoskeleton is abnormal, with an increased proportion of unbundled microtubules. Reducing PP2A enzymatic activity also leads to an increase in unbundled microtubules, an effect enhanced by reducing wrd levels. Hence, wrd promotes the function of PP2A and is required for normal cytoskeletal organization, synaptic growth, and synaptic function at the Drosophila NMJ.

Tuberous sclerosis complex regulates Drosophila neuromuscular junction growth via the TORC2/Akt pathway

Mutations in the tuberous sclerosis complex (TSC) are associated with various forms of neurodevelopmental disorders, including autism and epilepsy. The heterodimeric TSC complex, consisting of Tsc1 and Tsc2 proteins, regulates the activity of the TOR (target of rapamycin) complex via Rheb, a small GTPase. TOR, an atypical serine/threonine kinase, forms two distinct complexes TORC1 and TORC2. Raptor and Rictor serve as specific functional components of TORC1 and TORC2, respectively. Previous studies have identified Tsc1 as a regulator of hippocampal neuronal morphology and function via the TOR pathway, but it is unclear whether this is mediated via TORC1 or TORC2. In a genetic screen for aberrant synaptic growth at the neuromuscular junctions (NMJs) in Drosophila, we identified that Tsc2 mutants showed increased synaptic growth. Increased synaptic growth was also observed in rictor mutants, while raptor knockdown did not phenocopy the TSC mutant phenotype, suggesting that a novel role exists for TORC2 in regulating synapse growth. Furthermore, Tsc2 mutants showed a dramatic decrease in the levels of phosphorylated Akt, and interestingly, Akt mutants phenocopied Tsc2 mutants, leading to the hypothesis that Tsc2 and Akt might work via the same genetic pathway to regulate synapse growth. Indeed, transheterozygous analysis of Tsc2 and Akt mutants confirmed this hypothesis. Finally, our data also suggest that while overexpression of rheb results in aberrant synaptic overgrowth, the overgrowth might be independent of TORC2. Thus, we propose that at the Drosophila NMJ, TSC regulates synaptic growth via the TORC2-Akt pathway.

CK2α regulates the transcription of BRP in Drosophila.

Development and plasticity of synapses are brought about by a complex interplay between various signaling pathways. Typically, either changing the number of synapses or strengthening an existing synapse can lead to changes during synaptic plasticity. Altering the machinery that governs the exocytosis of synaptic vesicles, which primarily fuse at specialized structures known as active zones on the presynaptic terminal, brings about these changes. Although signaling pathways that regulate the synaptic plasticity from the postsynaptic compartments are well defined, the pathways that control these changes presynaptically are poorly described. In a genetic screen for synapse development in Drosophila, we found that mutations in CK2α lead to an increase in the levels of Bruchpilot (BRP), a scaffolding protein associated with the active zones. Using a combination of genetic and biochemical approaches, we found that the increase in BRP in CK2α mutants is largely due to an increase in the transcription of BRP. Interestingly, the transcripts of other active zone proteins that are important for function of active zones were also increased, while the transcripts from some other synaptic proteins were unchanged. Thus, our data suggest that CK2α might be important in regulating synaptic plasticity by modulating the transcription of BRP. Hence, we propose that CK2α is a novel regulator of the active zone protein, BRP, in Drosophila.