Iain M. Robinson

Independent Regulation of Synaptic Size and Activity by the Anaphase-Promoting Complex

Neuronal plasticity relies on tightly regulated control of protein levels at synapses. One mechanism to control protein abundance is the ubiquitin-proteasome degradation system. Recent studies have implicated ubiquitin-mediated protein degradation in synaptic development, function, and plasticity, but little is known about the regulatory mechanisms controlling ubiquitylation in neurons. In contrast, ubiquitylation has long been studied as a central regulator of the eukaryotic cell cycle. A critical mediator of cell-cycle transitions, the anaphase-promoting complex/cyclosome (APC/C), is an E3 ubiquitin ligase. Although the APC/C has been detected in several differentiated cell types, a functional role for the complex in postmitotic cells has been elusive. We describe a novel postmitotic role for the APC/C at Drosophila neuromuscular synapses: independent regulation of synaptic growth and synaptic transmission. In neurons, the APC/C controls synaptic size via a downstream effector Liprin-alpha; in muscles, the APC/C regulates synaptic transmission, controlling the concentration of a postsynaptic glutamate receptor.

Synaptotagmins I and IV promote transmitter release independently of Ca(2+) binding in the C(2)A domain.

At nerve terminals, a focal and transient increase in intracellular Ca2+ triggers the fusion of neurotransmitter-filled vesicles with the plasma membrane. The most extensively studied candidate for the Ca2+-sensing trigger is synaptotagmin I, whose Ca2+-dependent interactions with acidic phospholipids and syntaxin have largely been ascribed to its C2A domain, although the C2B domain also binds Ca2+ (refs 7, 8). Genetic tests of synaptotagmin I have been equivocal as to whether it is the Ca2+-sensing trigger of fusion. Synaptotagmin IV, a related isoform that does not bind Ca2+ in the C2A domain, might be an inhibitor of release. We mutated an essential aspartate of the Ca2+-binding site of the synaptotagmin I C2A domain and expressed it in Drosophila lacking synaptotagmin I. Here we show that, despite the disruption of the binding site, the Ca2+-dependent properties of transmission were not altered. Similarly, we found that synaptotagmin IV could substitute for synaptotagmin I. We conclude that the C2A domain of synaptotagmin is not required for Ca2+-dependent synaptic transmission, and that synaptotagmin IV promotes rather than inhibits transmission.

Dysregulation of ubiquitin homeostasis and β-catenin signaling promote spinal muscular atrophy

The autosomal recessive neurodegenerative disease spinal muscular atrophy (SMA) results from low levels of survival motor neuron (SMN) protein; however, it is unclear how reduced SMN promotes SMA development. Here, we determined that ubiquitin-dependent pathways regulate neuromuscular pathology in SMA. Using mouse models of SMA, we observed widespread perturbations in ubiquitin homeostasis, including reduced levels of ubiquitin-like modifier activating enzyme 1 (UBA1). SMN physically interacted with UBA1 in neurons, and disruption of Uba1 mRNA splicing was observed in the spinal cords of SMA mice exhibiting disease symptoms. Pharmacological or genetic suppression of UBA1 was sufficient to recapitulate an SMA-like neuromuscular pathology in zebrafish, suggesting that UBA1 directly contributes to disease pathogenesis. Dysregulation of UBA1 and subsequent ubiquitination pathways led to β-catenin accumulation, and pharmacological inhibition of β-catenin robustly ameliorated neuromuscular pathology in zebrafish, Drosophila, and mouse models of SMA. UBA1-associated disruption of β-catenin was restricted to the neuromuscular system in SMA mice; therefore, pharmacological inhibition of β-catenin in these animals failed to prevent systemic pathology in peripheral tissues and organs, indicating fundamental molecular differences between neuromuscular and systemic SMA pathology. Our data indicate that SMA-associated reduction of UBA1 contributes to neuromuscular pathogenesis through disruption of ubiquitin homeostasis and subsequent β-catenin signaling, highlighting ubiquitin homeostasis and β-catenin as potential therapeutic targets for SMA.

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.

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.

Pulsed laser imaging of rapid Ca2+ gradients in excitable cells

Excitable cells are thought to respond to action potentials by forming short lived and highly localized Ca2+ gradients near sites of Ca2+ entry or near the site of Ca2+ release by intracellular stores. However, conventional imaging techniques lack the spatial and temporal resolution to capture these gradients. Here we demonstrate the use of pulsed-laser microscopy to measure Ca2+ gradients with submicron spatial resolution and millisecond time resolution in two preparations where the Ca2+ signal is thought to be fast and highly localized: adrenal chromaffin cells, where the entry of Ca2+ through voltage dependent Ca2+ channels triggers exocytotic fusion; and skeletal muscle fibers, where intracellular Ca2+ release from the sarcoplasmic reticulum initiates contraction. In chromaffin cells, Ca2+ gradients developed over 10-100 ms and were initially restricted to discrete submembrane domains, or hot spots, before developing into complete rings of elevated Ca2+ concentration. In frog skeletal muscle large, short-lived (approximately 6 ms) Ca2+ gradients were observed within individual sarcomeres following induction of action potentials. The pulsed laser imaging approach permits, for the first time, the capture and critical examination of rapid Ca2+ signaling events such as those underlying excitation-secretion and excitation-contraction coupling.

Simultaneous capacitance and amperometric measurements of exocytosis: a comparison

We measured the exocytotic response induced by flash photolysis of caged compounds in isolated mast cells and chromaffin cells. Vesicle fusion was measured by monitoring the cell membrane capacitance. The release of vesicular contents was followed by amperometry. In response to a GTP gamma S stimulus we found that the time integral of the amperometric current could be superimposed on the capacitance trace. This shows that the integrated amperometric signal provides an alternative method of measuring the extent and kinetics of the secretory response. Very different results were obtained when photolysis of caged Ca2+ (DM-nitrophen) was used to stimulate secretion. In mast cells, there was an immediate, graded increase in membrane capacitance that was followed by step increases (indicative of granule fusion). During the initial phase of the capacitance increases, no release of oxidizable secretory products was detected. In chromaffin cells we also observed a considerable delay between increases in capacitance, triggered by uncaging Ca2+, and the release of oxidizable secretory products. Here we demonstrate that there can be large increases in the membrane capacitance of a secretory cell, triggered by flash photolysis of DM-nitrophen, which indicate events that are not due to the fusion of granules containing oxidizable substances. These results show that increases in capacitance that are not resolved as steps cannot be readily interpreted as secretory events unless they are confirmed independently.

Characterisation of Distinct Inositol 1,4,5-Trisphosphate-Sensitive and Caffeine-Sensitive Calcium Stores in Digitonin-Permeabilised Adrenal Chromaffin Cells

Abstract: The effect of inositol 1,4,5‐trisphosphate [Ins‐(1,4,5)P3] and caffeine on Ca2+ release from digitonin‐per‐meabilised bovine adrenal chromaffin cells was examined by using the Ca2+ indicator fura‐2 to monitor [Ca2+]. Permea‐bilised cells accumulated Ca2+ in the presence of ATP and addition of either Ins(1,4,5)P3 or caffeine released 17% or 40–50%, respectively, of the accumulated Ca2+, indicated by sustained rises in [Ca2+] in the cell suspension. Prior addition of Ins(l,4,5)P3 had no effect on the magnitude of the response to a subsequent addition of caffeine. The response to Ins(l,4,5)P3 was prevented by prior addition of caffeine or CaCl2, indicating that the Ins(l,4,5)P3 response was blocked by elevated [Ca2+]. The responses were essentially identical in the presence of the proton ionophore carbonyl cyanide m‐chlorophenylhydrazone, indicating that the Ca2+ release was not from mitochondria or secretory granules and that a proton gradient was not required for Ca2+ accumulation into the Ins(l,4,5)P3‐ or caffeine‐sensitive stores. Ca2+ release from the caffeine‐sensitive store was selectively blocked by ryano‐dine. The Ins(l,4,5)P3‐sensitive store was emptied by thap‐sigargin, which had no effect on caffeine responses. These data suggest that permeabilised chromaffin cells possess two distinct nonoverlapping Ca2+ stores sensitive to either Ins(1,4,5)P3 or caffeine and support previous conclusions that these stores possess different Ca2+‐ATPases.

Loss and gain of Drosophila TDP-43 impair synaptic efficacy and motor control leading to age-related neurodegeneration by loss-of-function phenotypes.

Cytoplasmic accumulation and nuclear clearance of TDP-43 characterize familial and sporadic forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, suggesting that either loss or gain of TDP-43 function, or both, cause disease formation. Here we have systematically compared loss- and gain-of-function of Drosophila TDP-43, TAR DNA Binding Protein Homolog (TBPH), in synaptic function and morphology, motor control, and age-related neuronal survival. Both loss and gain of TBPH severely affect development and result in premature lethality. TBPH dysfunction caused impaired synaptic transmission at the larval neuromuscular junction (NMJ) and in the adult. Tissue-specific knockdown together with electrophysiological recordings at the larval NMJ also revealed that alterations of TBPH function predominantly affect pre-synaptic efficacy, suggesting that impaired pre-synaptic transmission is one of the earliest events in TDP-43-related pathogenesis. Prolonged loss and gain of TBPH in adults resulted in synaptic defects and age-related, progressive degeneration of neurons involved in motor control. Toxic gain of TBPH did not downregulate or mislocalize its own expression, indicating that a dominant-negative effect leads to progressive neurodegeneration also seen with mutational inactivation of TBPH. Together these data suggest that dysfunction of Drosophila TDP-43 triggers a cascade of events leading to loss-of-function phenotypes whereby impaired synaptic transmission results in defective motor behavior and progressive deconstruction of neuronal connections, ultimately causing age-related neurodegeneration.

Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca2+ channel-vesicle coupling

Brain function relies on fast and precisely timed synaptic vesicle (SV) release at active zones (AZs). Efficacy of SV release depends on distance from SV to Ca2+ channel, but molecular mechanisms controlling this are unknown. Here we found that distances can be defined by targeting two unc-13 (Unc13) isoforms to presynaptic AZ subdomains. Super-resolution and intravital imaging of developing Drosophila melanogaster glutamatergic synapses revealed that the Unc13B isoform was recruited to nascent AZs by the scaffolding proteins Syd-1 and Liprin-α, and Unc13A was positioned by Bruchpilot and Rim-binding protein complexes at maturing AZs. Unc13B localized 120 nm away from Ca2+ channels, whereas Unc13A localized only 70 nm away and was responsible for docking SVs at this distance. Unc13Anull mutants suffered from inefficient, delayed and EGTA-supersensitive release. Mathematical modeling suggested that synapses normally operate via two independent release pathways differentially positioned by either isoform. We identified isoform-specific Unc13-AZ scaffold interactions regulating SV-Ca2+-channel topology whose developmental tightening optimizes synaptic transmission.