Mutations in dynamin-related protein result in gross changes in mitochondrial morphology and affect synaptic vesicle recycling at the Drosophila neuromuscular junction

Mitochondria are the primary source of ATP needed for the steps of the synaptic vesicle cycle. Dynamin-related protein (DRP) is involved in the fission of mitochondria and peroxisomes. To assess the role of mitochondria in synaptic function, we characterized a Drosophila DRP mutant combination that shows an acute temperature-sensitive paralysis. Sequencing of the mutant reveals a single amino acid change in the guanosine triphosphate hydrolysing domain (GTPase domain) of DRP. The synaptic mitochondria in these mutants are remarkably elongated, suggesting a role for DRP in mitochondrial fission in Drosophila. There is a loss of neuronal transmission at restrictive temperatures in electroretinogram (ERG) recordings. Like stress-sensitive B (sesB), a mitochondrial adenosine triphosphate (ATP) translocase mutant we studied earlier for its effects on synaptic vesicle recycling, an allele-specific reduction in the temperature of paralysis of Drosophila synaptic vesicle recycling mutant shibire was seen in the DRP mutant background. These data, in addition to depletion of vesicles observed in electron microscopic sections of photoreceptor synapses at restrictive temperatures, suggest a block in synaptic vesicle recycling due to reduced mitochondrial function.     

Key physiological parameters dictate triggering of activity-dependent bulk endocytosis in hippocampal synapses

To maintain neurotransmission in central neurons, several mechanisms are employed to retrieve synaptically exocytosed membrane. The two major modes of synaptic vesicle (SV) retrieval are clathrin-mediated endocytosis and activity-dependent bulk endocytosis (ADBE). ADBE is the dominant SV retrieval mode during intense stimulation, however the precise physiological conditions that trigger this mode are not resolved. To determine these parameters we manipulated rat hippocampal neurons using a wide spectrum of stimuli by varying both the pattern and duration of stimulation. Using live-cell fluorescence imaging and electron microscopy approaches, we established that stimulation frequency, rather than the stimulation load, was critical in the triggering of ADBE. Thus two hundred action potentials, when delivered at high frequency, were sufficient to induce near maximal bulk formation. Furthermore we observed a strong correlation between SV pool size and ability to perform ADBE. We also identified that inhibitory nerve terminals were more likely to utilize ADBE and had a larger SV recycling pool. Thus ADBE in hippocampal synaptic terminals is tightly coupled to stimulation frequency and is more likely to occur in terminals with large SV pools. These results implicate ADBE as a key modulator of both hippocampal neurotransmission and plasticity.     

Drosophila Amphiphysin is a Post-Synaptic Protein Required for Normal Locomotion but Not Endocytosis

Clathrin-mediated endocytosis is required to recycle synaptic vesicles for fast and efficient neurotransmission. Amphiphysins are thought to be multiprotein adaptors that may contribute to this process by bringing together many of the proteins required for endocytosis. Their in vivo function, however, has yet to be determined. Here, we show that the Drosophila genome encodes a single amphiphysin gene that is broadly expressed during development. We also show that, unlike its vertebrate counterparts, Drosophila Amphiphysin is enriched postsynaptically at the larval neuromuscular junction. To determine the role of Drosophila Amphiphysin, we also generated null mutants which are viable but give rise to larvae and adults with pronounced locomotory defects. Surprisingly, the locomotory defects cannot be accounted for by alterations in the morphology or physiology of the neuromuscular junction. Moreover, using stimulus protocols designed to test endocytosis under moderate and extreme vesicle cycling, we could not detect any defect in the neuromuscular junction of the amphiphysin mutant. Taken together, our findings suggest that Amphiphysin is not required for viability, nor is it absolutely required for clathrin-mediated endocytosis. However, Drosophila Amphiphysin function is required in both larvae and adults for normal locomotion.     

Dap160/intersectin acts as a stabilizing scaffold required for synaptic development and vesicle endocytosis

We describe the isolation of mutations in dynamin-associated protein 160 kDa (dap160), the Drosophila homolog of intersectin, a putative adaptor for proteins involved in endocytosis, cytoskeletal regulation, and signaling. We show that partial loss-of-function mutants display temperature-sensitive (ts) paralysis, whereas null mutants show ts defects in endocytosis. Loss-of-function mutants exhibit bouton overgrowth at larval neuromuscular junctions (NMJs), but evoked neurotransmission is normal. Mutant NMJs show a mild endocytic defect at 22 degrees C, which is strongly enhanced at 34 degrees C. The levels of dynamin, synaptojanin and endophilin are severely reduced in dap160 mutant NMJs, suggesting that Dap160 serves to stabilize an endocytic macromolecular complex. Electron microscopy reveals fewer vesicles, aberrant large vesicles, and an accumulation of endocytic intermediates at active and periactive zones in mutant terminals. Our data suggest that Dap160, like dynamin, is involved in synaptic vesicle retrieval at active and periactive zones.     

Similar oxysterols may lead to opposite effects on synaptic transmission: Olesoxime versus 5α-cholestan-3-one at the frog neuromuscular junction

Cholesterol oxidation products frequently have a high biological activity. In the present study, we have used microelectrode recording of end plate currents and FM-based optical detection of synaptic vesicle exo-endocytosis to investigate the effects of two structurally similar oxysterols, olesoxime (cholest-4-en-3-one, oxime) and 5ɑ-cholestan-3-one (5ɑCh3), on neurotransmission at the frog neuromuscular junction. Olesoxime is an exogenous, potentially neuroprotective, substance and 5ɑCh3 is an intermediate product in cholesterol metabolism, which is elevated in the case of cerebrotendinous xanthomatosis. We found that olesoxime slightly increased evoked neurotransmitter release in response to a single stimulus and significantly reduced synaptic depression during high frequency activity. The last effect was due to an increase in both the number of synaptic vesicles involved in exo-endocytosis and the rate of synaptic vesicle recycling. In contrast, 5ɑCh3 reduced evoked neurotransmitter release during the low- and high frequency synaptic activities. The depressant action of 5ɑCh3 was associated with a reduction in the number of synaptic vesicles participating in exo- and endocytosis during high frequency stimulation, without a change in rate of the synaptic vesicle recycling. Of note, olesoxime increased the staining of synaptic membranes with the B-subunit of cholera toxin and the formation of fluorescent ganglioside GM1 clusters, and decreased the fluorescence of 22-NBD-cholesterol, while 5ɑCh3 had the opposite effects, suggesting that the two oxysterols have different effects on lipid raft stability. Taken together, these data show that these two structurally similar oxysterols induce marked different changes in neuromuscular transmission which are related with the alteration in synaptic vesicle cycle.     

Drosophila CAPS is an essential gene that regulates dense-core vesicle release and synaptic vesicle fusion

Calcium-activated protein for secretion (CAPS) is proposed to play an essential role in Ca2+-regulated dense-core vesicle exocytosis in vertebrate neuroendocrine cells. Here we report the cloning, mutation, and characterization of the Drosophila ortholog (dCAPS). Null dCAPS mutants display locomotory deficits and complete embryonic lethality. The mutant NMJ reveals a 50% loss in evoked glutamatergic transmission, and an accumulation of synaptic vesicles at active zones. Importantly, dCAPS mutants display a highly specific 3-fold accumulation of dense-core vesicles in synaptic terminals, which was not observed in mutants that completely arrest synaptic vesicle exocytosis. Targeted transgenic CAPS expression in identified motoneurons fails to rescue dCAPS neurotransmission defects, demonstrating a cell nonautonomous role in synaptic vesicle fusion. We conclude that dCAPS is required for dense-core vesicle release and that a dCAPS-dependent mechanism modulates synaptic vesicle release at glutamatergic synapses.     

Temporal requirements of the fragile X mental retardation protein in the regulation of synaptic structure

Fragile X syndrome (FraX), caused by the loss-of-function of one gene (FMR1), is the most common inherited form of both mental retardation and autism spectrum disorders. The FMR1 product (FMRP) is an mRNA-binding translation regulator that mediates activity-dependent control of synaptic structure and function. To develop any FraX intervention strategy, it is essential to define when and where FMRP loss causes the manifestation of synaptic defects, and whether the reintroduction of FMRP can restore normal synapse properties. In the Drosophila FraX model, dFMRP loss causes neuromuscular junction (NMJ) synapse over-elaboration (overgrowth, overbranching, excess synaptic boutons), accumulation of development-arrested satellite boutons, and altered neurotransmission. We used the Gene-Switch method to conditionally drive dFMRP expression to define the spatiotemporal requirements in synaptic mechanisms. Constitutive induction of targeted neuronal dFMRP at wild-type levels rescues all synaptic architectural defects in Drosophila Fmr1 (dfmr1)-null mutants, demonstrating a presynaptic requirement for synapse structuring. By contrast, presynaptic dFMRP expression does not ameliorate functional neurotransmission defects, indicating a postsynaptic dFMRP requirement. Strikingly, targeted early induction of dFMRP effects nearly complete rescue of synaptic structure defects, showing a primarily early-development role. In addition, acute dFMRP expression at maturity partially alleviates dfmr1-null defects, although rescue is not as complete as either early or constitutive dFMRP expression, showing a modest capacity for late-stage structural plasticity. We conclude that dFMRP predominantly acts early in synaptogenesis to modulate architecture, but that late dFMRP introduction at maturity can weakly compensate for early absence of dFMRP function.     

Impairment of central pattern generation in <Emphasis Type="Italic">Drosophila</Emphasis> cysteine string protein mutants

The cysteine string proteins are integral synaptic vesicle proteins, critical for fast, Ca2+ -regulated exocytosis. Drosophila larvae with null mutations for the cysteine string protein (csp) gene have temperature-sensitive impairments of neurotransmission and presynaptic calcium removal at the neuromuscular junction. Using the larval Drosophila preparation to examine central pattern generation, we characterized the temperature sensitivity of locomotor patterns in wildtype and csp mutant larvae. Intraburst frequency of motoneuronal activity reached 100 Hz and was sufficiently high to rescue the temperature-sensitive synaptic failure in the mutant. Nevertheless, we show that deletion of the csp gene resulted in a severe deficiency in the generation of coordinated larval motor rhythms. Csp mutants that could generate patterned motor activity had slower, poorly coordinated rhythms with altered temperature sensitivity. We conclude that the temperature-sensitive paralysis characteristic of csp mutants is not a direct result of synaptic failure at neuromuscular junctions, as might be expected, but is the result of a failure of locomotor circuit operation at a higher integrative level.     

Modulation of Synaptic Vesicle Exocytosis in Muscle-Dependent Long-Term Depression at the Amphibian Neuromuscular Junction

We have labeled recycling synaptic vesicles at the somatic Bufo marinus neuromuscular junction with the styryl dye FM2-10 and provide direct evidence for refractoriness of exocytosis associated with a muscle activity-dependent form of long-term depression (LTD) at this synapse. FM2-10 dye unloading experiments demonstrated that the rate of vesicle exocytosis from the release ready pool (RRP) of vesicles was more than halved in the LTD (induced by 20 min of low frequency stimulation). Recovery from LTD, observed as a partial recovery of nerve-evoked muscle twitch amplitude, was accompanied by partial recovery of the refractoriness of RRP exocytosis. Unexpectedly, paired pulse plasticity, another routinely used indicator of presynaptic forms of synaptic plasticity, was unchanged in the LTD. We conclude that the LTD induces refractoriness of the neuromuscular vesicle release machinery downstream of presynaptic calcium entry.     

Loss of skywalker reveals synaptic endosomes as sorting stations for synaptic vesicle proteins

Exchange of proteins at sorting endosomes is not only critical to numerous signaling pathways but also to receptor-mediated signaling and to pathogen entry into cells; however, how this process is regulated in synaptic vesicle cycling remains unexplored. In this work, we present evidence that loss of function of a single neuronally expressed GTPase activating protein (GAP), Skywalker (Sky) facilitates endosomal trafficking of synaptic vesicles at Drosophila neuromuscular junction boutons, chiefly by controlling Rab35 GTPase activity. Analyses of genetic interactions with the ESCRT machinery as well as chimeric ubiquitinated synaptic vesicle proteins indicate that endosomal trafficking facilitates the replacement of dysfunctional synaptic vesicle components. Consequently, sky mutants harbor a larger readily releasable pool of synaptic vesicles and show a dramatic increase in basal neurotransmitter release. Thus, the trafficking of vesicles via endosomes uncovered using sky mutants provides an elegant mechanism by which neurons may regulate synaptic vesicle rejuvenation and neurotransmitter release.     

Essential role of endophilin A in synaptic vesicle budding at the Drosophila neuromuscular junction

We characterized Drosophila endophilin A (D-endoA), and generated and analysed D-endoA mutants. Like its mammalian homologue, D-endoA exhibits lysophosphatidic acid acyl transferase activity and contains a functional SH3 domain. D-endoA is recruited to the sites of endocytosis, as revealed by immunocytochemistry of the neuromuscular junction (NMJ) of mutant L3 larvae carrying the temperature-sensitive allele of dynamin, shibire. D-endoA null mutants show severe defects in motility and die at the early L2 larval stage. Mutants with reduced D-endoA levels exhibit a range of defects of synaptic vesicle endocytosis, as observed at L3 larvae NMJs using FM1-43 uptake and electron microscopy. NMJs with an almost complete loss of synaptic vesicles did not show an accumulation of intermediates of the budding process, whereas NMJs with only slightly reduced levels of synaptic vesicles showed a striking increase in early-stage, but not late-stage, budding intermediates at the plasma membrane. Together with results of previous studies, these observations indicate that endophilin A is essential for synaptic vesicle endocytosis, being required from the onset of budding until fission.     

A temperature-sensitive allele of Drosophila sesB reveals acute functions for the mitochondrial adenine nucleotide translocase in synaptic transmission and dynamin regulation

Rapidly reversible, temperature-sensitive (ts) paralytic mutants of Drosophila have been useful in delineating immediate in vivo functions of molecules involved in synaptic transmission. Here we report isolation and characterization of orangi (org), an enhancer of shibire (shi), a ts paralytic mutant in Drosophila dynamin. org is an allele of the stress sensitive B (sesB) locus that encodes a mitochondrial adenine nucleotide translocase (ANT) and results in a unique ts paralytic behavior that is accompanied by a complete loss of synaptic transmission in the visual system. sesB(org) reduces the restrictive temperature for all shi(ts) alleles tested except for shi(ts1). This characteristic allele-specific interaction of sesB(org) with shi is shared by abnormal wing discs (awd), a gene encoding nucleoside diphosphate kinase (NDK). sesB(org) shows independent synergistic interactions, an observation that is consistent with a shared pathway by which org and awd influence shi function. Genetic and electrophysiological analyses presented here, together with the observation that the sesB(org) mutation reduces biochemically assayed ANT activity, suggest a model in which a continuous mitochondrial ANT-dependent supply of ATP is required to sustain NDK-dependent activation of presynaptic dynamin during a normal range of synaptic activity.     

Olesoxime,a cholesterol-like neuroprotectant restrains synaptic vesicle exocytosis in the mice motor nerve terminals: Possible role of VDACs

Olesoxime is a cholesterol-like neuroprotective compound that targets to mitochondrial voltage dependent anion channels (VDACs). VDACs were also found in the plasma membrane and highly expressed in the presynaptic compartment. Here, we studied the effects of olesoxime and VDAC inhibitors on neurotransmission in the mouse neuromuscular junction. Electrophysiological analysis revealed that olesoxime suppressed selectively evoked neurotransmitter release in response to a single stimulus and 20 Hz activity. Also olesoxime decreased the rate of FM1–43 dye loss (an indicator of synaptic vesicle exocytosis) at low frequency stimulation and 20 Hz. Furthermore, an increase in extracellular Cl⁻ enhanced the action of olesoxime on the exocytosis and olesoxime increased intracellular Cl⁻ levels. The effects of olesoxime on the evoked synaptic vesicle exocytosis and [Cl⁻]_i were blocked by membrane-permeable and impermeable VDAC inhibitors. Immunofluorescent labeling pointed on the presence of VDACs on the synaptic membranes. Rotenone-induced mitochondrial dysfunction perturbed the exocytotic release of FM1–43 and cell-permeable VDAC inhibitor (but not olesoxime or impermeable VDAC inhibitor) partially mitigated the rotenone-driven alterations in the FM1–43 unloading and mitochondrial superoxide production. Thus, olesoxime restrains neurotransmission by acting on plasmalemmal VDACs whose activation can limit synaptic vesicle exocytosis probably via increasing anion flux into the nerve terminals.     

Dominant-negative NSF2 disrupts the structure and function of Drosophila neuromuscular synapses

N-ethylmaleimide sensitive fusion protein (NSF) is an ATPase necessary for vesicle trafficking, including exocytosis. Current models hold that NSF is required in a step that readies vesicles for fusion by disassembling postfusion SNARE protein complexes allowing them to participate in further rounds of vesicle cycling. Whereas most organisms have only one NSF isoform, Drosophila has two. dNSF1 is the predominant functional isoform in the adult nervous system. Conditional mutations in the dNSF1 gene, comatose, are paralytic and lead to disruption of synaptic transmission and the rapid accumulation of SNARE complexes in adult flies. This isoform is not required for synaptic transmission in larvae. In contrast, dNSF2 is important at earlier developmental stages, and its broad expression indicates its importance in neural and non-neural tissues alike. To study dNSF2, and to circumvent the lethality of dNSF2 null mutants, we have constructed transgenic flies carrying a dominant negative form of dNSF2. When this construct was expressed in neurons we observed suppression of synaptic transmission, activity-dependent fatigue of transmitter release, and a reduction in the number of releasable vesicles. However, we unexpectedly found that there was no accumulation of SNARE complexes accompanying these physiological phenotypes. Intriguingly, we also found that expression of mutant dNSF2 induced pronounced overgrowth of the neuromuscular junction and some misrouting of axons. These results support the idea that dNSF2 has multiple roles in cellular function and adds that not all of its functions require disassembly of the SNARE complex.     

Synaptojanin is recruited by endophilin to promote synaptic vesicle uncoating

We describe the isolation and characterization of Drosophila synaptojanin (synj) mutants. synj encodes a phosphatidylinositol phosphatase involved in clathrin-mediated endocytosis. We show that Synj is specifically localized to presynaptic terminals and is associated with synaptic vesicles. The electrophysiological and ultrastructural defects observed in synj mutants are strikingly similar to those found in endophilin mutants, and Synj and Endo colocalize and interact biochemically. Moreover, synj; endo double mutant synaptic terminals exhibit properties that are very similar to terminals of each single mutant, and overexpression of Endophilin can partially rescue the functional defects in partial loss-of-function synj mutants. Interestingly, Synj is mislocalized and destabilized at synapses devoid of Endophilin, suggesting that Endophilin recruits and stabilizes Synj on newly formed vesicles to promote vesicle uncoating. Our data also provide further evidence that kiss-and-run is able to maintain neurotransmitter release when synapses are not extensively challenged.     

The use of pHluorins for optical measurements of presynaptic activity

Genetically encoded reporters for optical measurements of presynaptic activity hold significant promise for measurements of neurotransmission within intact or semi-intact neuronal networks. We have characterized pH-sensitive green fluorescent protein-based sensors (pHluorins) of synaptic vesicle cycling at nerve terminals. pHluorins have a pK approximately 7.1, which make them ideal for tracking synaptic vesicle lumen pH upon cycling through the plasma membrane during action potentials. A theoretical analysis of the expected signals using this approach and guidelines for future reporter development are provided.     

Localization and Mobility of Synaptic Vesicles in Myosin VI Mutants of Drosophila

Background

At the Drosophila neuromuscular junction (NMJ), synaptic vesicles are mobile; however, the mechanisms that regulate vesicle traffic at the nerve terminal are not fully understood. Myosin VI has been shown to be important for proper synaptic physiology and morphology at the NMJ, likely by functioning as a vesicle tether. Here we investigate vesicle dynamics in Myosin VI mutants of Drosophila.

Results

In Drosophila, Myosin VI is encoded by the gene, jaguar (jar). To visualize active vesicle cycling we used FM dye loading and compared loss of function alleles of jar with controls. These studies revealed a differential distribution of vesicles at the jar mutant nerve terminal, with the newly endocytosed vesicles observed throughout the mutant boutons in contrast to the peripheral localization visualized at control NMJs. This finding is consistent with a role for Myosin VI in restraining vesicle mobility at the synapse to ensure proper localization. To further investigate regulation of vesicle dynamics by Myosin VI, FRAP analysis was used to analyze movement of GFP-labeled synaptic vesicles within individual boutons. FRAP revealed that synaptic vesicles are moving more freely in the jar mutant boutons, indicated by changes in initial bleach depth and rapid recovery of fluorescence following photobleaching.

Conclusion

This data provides insights into the role for Myosin VI in mediating synaptic vesicle dynamics at the nerve terminal. We observed mislocalization of actively cycling vesicles and an apparent increase in vesicle mobility when Myosin VI levels are reduced. These observations support the notion that a major function of Myosin VI in the nerve terminal is tethering synaptic vesicles to proper sub-cellular location within the bouton.     

Vps74 gives phosphatase directions

Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. In this paper, we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, we show that synaptic vesicle-associated proteins were younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, we find that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. Our findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans.     

Vesicle dynamics: how synaptic proteins regulate different modes of neurotransmission

Central synapses operate neurotransmission in several modes: synchronous/fast neurotransmission (neurotransmitters release is tightly coupled to action potentials and fast), asynchronous neurotransmission (neurotransmitter release is slower and longer lasting), and spontaneous neurotransmission (where small amounts of neurotransmitter are released without being evoked by an action potential). A substantial body of evidence from the past two decades suggests that seemingly identical synaptic vesicles possess distinct propensities to fuse, thus selectively serving different modes of neurotransmission. In efforts to better understand the mechanism(s) underlying the different modes of synaptic transmission, many research groups found that synaptic vesicles used in different modes of neurotransmission differ by a number of synaptic proteins. Synchronous transmission with higher temporal fidelity to stimulation seems to require synaptotagmin 1 and complexin for its Ca²⁺ sensitivity, RIM proteins for closer location of synaptic vesicles (SV) to the voltage operated calcium channels (VGCC), and dynamin for SV retrieval. Asynchronous release does not seem to require functional synaptotagmin 1 as a calcium sensor or complexins, but the activity of dynamin is indispensible for its maintenance. On the other hand, the control of spontaneous neurotransmission remains less clear as deleting a number of essential synaptic proteins does not abolish this type of synaptic vesicle fusion. VGCC distance from the SV seems to have little control on spontaneous transmission, while there is an involvement of functional synaptic proteins including synaptotagmins and complexin. Recently, presynaptic deficits have been proposed to contribute to a number of pathological conditions including cognitive and mental disorders. In this review, we evaluate recent advances in understanding the regulatory mechanisms of synaptic vesicle dynamics and in understanding how different molecular substrates maintain selective modes of neurotransmission. We also highlight the implications of these studies in understanding pathological conditions.     

Alterations in transmission, vesicle dynamics, and transmitter release machinery at NCAM-deficient neuromuscular junctions.

Although functional neuromuscular junctions (NMJs) form in NCAM-deficient mice, they exhibit multiple alterations in presynaptic organization and function. Profound depression and unusual periodic total transmission failures with repetitive stimulation point to a defect in vesicle mobilization/cycling, and these defects were mimicked in (+/+) NMJs by inhibitors of myosin light chain kinase, known to affect vesicle mobilization. Two separate release mechanisms, utilizing different endocytic machinery and Ca(2+) channels, were shown to coexist in (-/-) terminals, with the mature process targeted to presynaptic membrane opposed to muscle, and an abnormally retained immature process targeted to the remainder of the presynaptic terminal and axon. Thus, NCAM plays a critical and heretofore unsuspected role in the molecular organization of the presynaptic NMJ.