Synaptotagmin is a synaptic vesicle protein that is postulated to be the Ca(2+) sensor for fast, evoked neurotransmitter release. Deleting the gene for synaptotagmin (syt(null)) strongly suppresses synaptic transmission in every species examined, showing that synaptotagmin is central in the synaptic vesicle cycle. The cytoplasmic region of synaptotagmin contains two C(2) domains, C(2)A and C(2)B. Five, highly conserved, acidic residues in both the C(2)A and C(2)B domains of synaptotagmin coordinate the binding of Ca(2+) ions, and biochemical studies have characterized several in vitro Ca(2+)-dependent interactions between synaptotagmin and other nerve terminal molecules. But there has been no direct evidence that any of the Ca(2+)-binding sites within synaptotagmin are required in vivo. Here we show that mutating two of the Ca(2+)-binding aspartate residues in the C(2)B domain (D(416,418)N in Drosophila) decreased evoked transmitter release by >95%, and decreased the apparent Ca(2+) affinity of evoked transmitter release. These studies show that the Ca(2+)-binding motif of the C(2)B domain of synaptotagmin is essential for synaptic transmission.

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 Ca(2+) 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 Ca(2+) channels, whereas Unc13A localized only 70 nm away and was responsible for docking SVs at this distance. Unc13A(null) 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-Ca(2+)-channel topology whose developmental tightening optimizes synaptic transmission.

Amphiphysins 1 and 2 are enriched in the mammalian brain and are proposed to recruit dynamin to sites of endocytosis. Shorter amphiphysin 2 splice variants are also found ubiquitously, with an enrichment in skeletal muscle. At the Drosophila larval neuromuscular junction, amphiphysin is localized postsynaptically and amphiphysin mutants have no major defects in neurotransmission; they are also viable, but flightless. Like mammalian amphiphysin 2 in muscles, Drosophila amphiphysin does not bind clathrin, but can tubulate lipids and is localized on T-tubules. Amphiphysin mutants have a novel phenotype, a severely disorganized T-tubule/sarcoplasmic reticulum system. We therefore propose that muscle amphiphysin is not involved in clathrin-mediated endocytosis, but in the structural organization of the membrane-bound compartments of the excitation-contraction coupling machinery of muscles.

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 SMAlike 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.

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.

At nerve terminals, a focal and transient increase in intracellular Ca(2+) triggers the fusion of neurotransmitter-filled vesicles with the plasma membrane. The most extensively studied candidate for the Ca(2+)-sensing trigger is synaptotagmin I, whose Ca(2+)-dependent interactions with acidic phospholipids and syntaxin have largely been ascribed to its C(2)A domain, although the C(2)B domain also binds Ca(2+) (refs 7, 8). Genetic tests of synaptotagmin I have been equivocal as to whether it is the Ca(2+)-sensing trigger of fusion. Synaptotagmin IV, a related isoform that does not bind Ca(2+) in the C(2)A domain, might be an inhibitor of release. We mutated an essential aspartate of the Ca(2+)-binding site of the synaptotagmin I C(2)A domain and expressed it in Drosophila lacking synaptotagmin I. Here we show that, despite the disruption of the binding site, the Ca(2+)-dependent properties of transmission were not altered. Similarly, we found that synaptotagmin IV could substitute for synaptotagmin I. We conclude that the C(2)A domain of synaptotagmin is not required for Ca(2+)-dependent synaptic transmission, and that synaptotagmin IV promotes rather than inhibits transmission.

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.

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.