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Despite decades of intense study, the molecular basis of asynchronous neurotransmitter release remains enigmatic. Synaptotagmin (syt) 7 and Doc2 have both been proposed as Ca2+ sensors that trigger this mode of exocytosis, but conflicting findings have led to controversy. Here, we demonstrate that at excitatory mouse hippocampal synapses, Doc2α is the major Ca2+ sensor for asynchronous release, while syt7 supports this process through activity-dependent docking of synaptic vesicles. In synapses lacking Doc2α, asynchronous release after single action potentials is strongly reduced, while deleting syt7 has no effect. However, in the absence of syt7, docked vesicles cannot be replenished on millisecond timescales. Consequently, both synchronous and asynchronous release depress from the second pulse onward during repetitive activity. By contrast, synapses lacking Doc2α have normal activity-dependent docking, but continue to exhibit decreased asynchronous release after multiple stimuli. Moreover, disruption of both Ca2+ sensors is non-additive. These findings result in a new model whereby syt7 drives activity-dependent docking, thus providing synaptic vesicles for synchronous (syt1) and asynchronous (Doc2 and other unidentified sensors) release during ongoing transmission.

Abstract Compensatory endocytosis keeps the membrane surface area of secretory cells constant following exocytosis. At chemical synapses, clathrin-independent ultrafast endocytosis maintains such homeostasis. This endocytic pathway is temporally and spatially coupled to exocytosis; it initiates within 50 ms at the region immediately next to the active zone where vesicles fuse. However, the coupling mechanism is unknown. Here, we demonstrate that filamentous actin is organized as a ring, surrounding the active zone at mouse hippocampal synapses. Assuming the membrane area conservation is due to this actin ring, our theoretical model suggests that flattening of fused vesicles exerts lateral compression in the plasma membrane, resulting in rapid formation of endocytic pits at the border between the active zone and the surrounding actin-enriched region. Consistent with model predictions, our data show that ultrafast endocytosis requires sufficient compression by exocytosis of multiple vesicles and does not initiate when actin organization is disrupted, either pharmacologically or by ablation of the actin-binding protein Epsin1. Our work suggests that membrane mechanics underlie the rapid coupling of exocytosis to endocytosis at synapses.

Existing optogenetic silencing methods affect membrane potential, biochemistry or protein integrity. Here, the authors demonstrate an approach for silencing synaptic transmission that combines fast activation and reversibility, by using nondisruptive, reversible, light-evoked clustering of synaptic vesicles, which they validate in Caenorhabditis elegans, zebrafish, and murine cell culture.

Approaches for manipulating individual organelles are important for learning more about their functions. Here the authors report a tool utilising phospholipase A/acyltransferases (PLAATs) for rapid defunctionalisation of organelles through remodelling of the membrane phospholipids.

Activation of voltage-gated calcium channels at presynaptic terminals leads to local increases in calcium and the fusion of synaptic vesicles containing neurotransmitter. Presynaptic output is a function of the density of calcium channels, the dynamic properties of the channel, the distance to docked vesicles, and the release probability at the docking site. We demonstrate that at Caenorhabditis elegans neuromuscular junctions two different classes of voltage-gated calcium channels, CaV2 and CaV1, mediate the release of distinct pools of synaptic vesicles. CaV2 channels are concentrated in densely packed clusters ~250 nm in diameter with the active zone proteins Neurexin, α-Liprin, SYDE, ELKS/CAST, RIM-BP, α-Catulin, and MAGI1. CaV2 channels are colocalized with the priming protein UNC-13L and mediate the fusion of vesicles docked within 33 nm of the dense projection. CaV2 activity is amplified by ryanodine receptor release of calcium from internal stores, triggering fusion up to 165 nm from the dense projection. By contrast, CaV1 channels are dispersed in the synaptic varicosity, and are colocalized with UNC-13S. CaV1 and ryanodine receptors are separated by just 40 nm, and vesicle fusion mediated by CaV1 is completely dependent on the ryanodine receptor. Distinct synaptic vesicle pools, released by different calcium channels, could be used to tune the speed, voltage-dependence, and quantal content of neurotransmitter release.

Abstract Background We recently reported a new absorbed dose conversion method, RAP (RAtio of Pharmacokinetics), for 211At-meta-astatobenzylguanidine (211At-MABG) using a single biodistribution measurement, the percent injected dose/g. However, there were some mathematical ambiguities in determining the optimal timing of a single measurement of the percent injected dose/g. Thus, we aimed to mathematically reconstruct the RAP method and to examine the optimal timing of a single measurement. Methods We derived a new formalism of the RAP dose conversion method at time t. In addition, we acquired a formula to determine the optimal timing of a single measurement of the percent injected dose/g, assuming the one-compartment model for biological clearance. Results We investigated the new formalism’s performance using a representative RAP coefficient with radioactive decay weighting. Dose conversions by representative RAP coefficients predicted the true [211At]MABG absorbed doses with an error of 10% or less. The inverses of the representative RAP coefficients plotted at 4 h post-injection, which was the optimal timing reported in the previous work, were very close to the new inverses of the RAP coefficients 4 h post-injection. Next, the behavior of the optimal timing was analyzed by radiolabeled compounds with physical half-lives of 7.2 h and 10 d on various biological clearance half-lives. Behavior maps of optimal timing showed a tendency to converge to a constant value as the biological clearance half-life of a target increased. The areas of optimal timing for both compounds within a 5% or 10% prediction error were distributed around the optimal timing when the biological clearance half-life of a target was equal to that of the reference. Finally, an example of RAP dose conversion was demonstrated for [211At]MABG. Conclusions The RAP dose conversion method renovated by the new formalism was able to estimate the [211At]MABG absorbed dose using a similar pharmacokinetics, such as [131I]MIBG. The present formalism revealed optimizing imaging time points on absorbed dose conversion between two radiopharmaceuticals. Further analysis and clinical data will be needed to elucidate the validity of a behavior map of the optimal timing of a single measurement for targeted alpha-nuclide therapy.

Haematopoietic insufficiency is the treatment target of lower-risk myelodysplastic syndrome (MDS). Although erythropoiesis-stimulating agents (ESAs) are generally effective for treating anaemia, resistance can develop. Hypoxia-inducible factor-prolyl hydroxylase (HIF-PH) improves renal anaemia by promoting endogenous erythropoietin production and normalizing iron metabolism. HIF-PH inhibitors could be used to treat MDS, but their efficacy and safety have not been studied. A 78-year-old female patient with essential thrombocythemia gradually developed anaemia and was diagnosed with therapy-related MDS 4 years later. The anaemia temporarily improved with ESAs, but the patient became transfusion dependent. At the same time, anaemia and chronic renal failure due to nephrosclerosis progressed, and the patient was diagnosed with MDS with renal anaemia. After switching from ESAs to roxadustat, an HIF-PH inhibitor, anaemia improved, and the patient was no longer transfusion dependent. No progression of the underlying disease or any adverse events was observed 4 months after initiating roxadustat.

For the mass production of astatine-211, a promising radiopharmaceutical for cancer treatment, we have proposed the innovative “Liquid Bismuth Target System.” The target window in this system must be made from a material that resists the highly corrosive liquid bismuth environment. To meet this requirement, a promising target window material was selected in corrosion experiments performed in stagnant liquid bismuth. Based on knowledge of corrosion in liquid lead-bismuth eutectic gained during the development of fast reactors and accelerator-driven subcritical systems, FeCrMo alloy, FeCrAl alloy and austenitic stainless steel (as a reference) were selected as the specimen materials. Experiments were carried out under saturated dissolved oxygen and low oxygen conditions, and the corrosion behaviors of the specimens were evaluated, mainly by scanning electron microscopy. The FeCrAl alloy exhibited the most excellent corrosion resistance, followed by FeCrMo alloy. Both materials are suitable candidates for the target window. Although austenitic stainless steel was less corrosion resistant than the former two materials, it is a likely applicable for the target window under appropriately limited operation conditions (such as irradiation current and exposure time) of the liquid bismuth target system.

Action potentials induce synchronous and asynchronous release of neurotransmitters. Here, the authors show that the synchronous and asynchronous release sites are aligned with AMPARs and NMDARs, respectively, in mouse hippocampal synapses. This organization allows efficient activation of NMDARs.

Synaptotagmin 7 (SYT7) has emerged as a key regulator of presynaptic function, but its localization and precise role in the synaptic vesicle cycle remain the subject of debate. Here, we used iGluSnFR to optically interrogate glutamate release, at the single-bouton level, in SYT7KO-dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired-pulse facilitation, and synaptic vesicle replenishment and found that SYT7 contributes to each of these processes to different degrees. ‘Zap-and-freeze’ electron microscopy revealed that a loss of SYT7 diminishes docking of synaptic vesicles after a stimulus and inhibits the recovery of depleted synaptic vesicles after a stimulus train. SYT7 supports these functions from the axonal plasma membrane, where its localization and stability require both γ-secretase-mediated cleavage and palmitoylation. In summary, SYT7 is a peripheral membrane protein that controls multiple modes of synaptic vesicle (SV) exocytosis and plasticity, in part, through enhancing activity-dependent docking of SVs.

The structural features of a synapse help determine its function. Synapses are extremely small and tightly packed with vesicles and other organelles. Visualizing synaptic structure requires imaging by electron microscopy, and the features in micrographs must be quantified, a process called morphometry. Three parameters are typically assessed from each specimen: (1) the sizes of individual vesicles and organelles; (2) the absolute number and densities of organelles; and (3) distances between organelles and key features at synapses, such as active zone membranes and dense projections. For data to be meaningful, the analysis must be repeated from hundreds to thousands of images from several biological replicates, a daunting task. Here we report a custom computer program to analyze key structural features of synapses: SynapsEM. In short, we developed ImageJ/Fiji macros to record x,y-coordinates of segmented structures. The coordinates are then exported as text files. Independent investigators can reload the images and text files to reexamine the segmentation using ImageJ. The Matlab program then calculates and reports key synaptic parameters from the coordinates. Since the values are calculated from coordinates, rather than measured from each micrograph, other parameters such as locations of docked vesicles relative to the center of an active zone can be extracted in Matlab by additional scripting. Thus, this program can accelerate the morphometry of synapses and promote a more comprehensive analysis of synaptic ultrastructure.

Dynamin-related protein 1 (Drp1) divides mitochondria as a mechano-chemical GTPase. However, the function of Drp1 beyond mitochondrial division is largely unknown. Multiple Drp1 isoforms are produced through mRNA splicing. One such isoform, Drp1ABCD, contains all four alternative exons and is specifically expressed in the brain. Here, we studied the function of Drp1ABCD in mouse neurons in both culture and animal systems using isoform-specific knockdown by shRNA and isoform-specific knockout by CRISPR/Cas9. We found that the expression of Drp1ABCD is induced during postnatal brain development. Drp1ABCD is enriched in dendritic spines and regulates postsynaptic clathrin-mediated endocytosis by positioning the endocytic zone at the postsynaptic density, independently of mitochondrial division. Drp1ABCD loss promotes the formation of ectopic dendrites in neurons and enhanced sensorimotor gating behavior in mice. These data reveal that Drp1ABCD controls postsynaptic endocytosis, neuronal morphology and brain function.

L-type amino acid transporter 1 (LAT1) might be a useful target for tumor therapy since it is highly expressed in various types of cancers. We previously developed an astatine-211 (211At)-labeled amino acid derivative, 2-211At-astato-α-methyl-L-phenylalanine (2-211At-AAMP), and demonstrated its therapeutic potential for LAT1-positive cancers. However, the therapeutic effect of 2-211At-AAMP was insufficient, probably due to its low tumor retention. The preloading of probenecid, an organic anion transporter inhibitor, can delay the clearance of some amino acid tracers from the blood and consequently increase their accumulation in tumors. In this study, we evaluated the effect of probenecid preloading on the biodistribution and therapeutic effect of 2-211At-AAMP in mice. In biodistribution studies, the blood radioactivity of 2-211At-AAMP significantly increased with probenecid preloading. Consequently, the accumulation of 2-211At-AAMP in tumors was significantly higher with probenecid than without probenecid loading. In a therapeutic study, tumor growth was suppressed by 2-211At-AAMP with probenecid, and the tumor volume was significantly lower in the treatment group than in the untreated control group from day 2 to day 30 (end of the follow-up period) after treatment. These results indicate that probenecid loading could improve the therapeutic effect of 2-211At-AAMP by increasing its accumulation in tumors.

(1) Background: Deferoxamine B (DFO) is the most widely used chelator for labeling of zirconium-89 (89Zr) to monoclonal antibody (mAb). Despite the remarkable developments of the clinical 89Zr-immuno-PET, chemical species and stability constants of the Zr-DFO complexes remain controversial. The aim of this study was to re-evaluate their stability constants by identifying species of Zr-DFO complexes and demonstrate that the stability constants can estimate radiochemical yield (RCY) and chelator-to-antibody ratio (CAR). (2) Methods: Zr-DFO species were determined by UV and ESI-MS spectroscopy. Stability constants and speciation of the Zr-DFO complex were redetermined by potentiometric titration. Complexation inhibition of Zr-DFO by residual impurities was investigated by competition titration. (3) Results: Unknown species, ZrHqDFO2, were successfully detected by nano-ESI-Q-MS analysis. We revealed that a dominant specie under radiolabeling condition (pH 7) was ZrHDFO, and its stability constant (logβ111) was 49.1 ± 0.3. Competition titration revealed that residual oxalate inhibits Zr-DFO complex formation. RCYs in different oxalate concentration (0.1 and 0.04 mol/L) were estimated to be 86% and >99%, which was in good agreement with reported results (87%, 97%). (4) Conclusion: This study succeeded in obtaining accurate stability constants of Zr-DFO complexes and estimating RCY and CAR from accurate stability constants established in this study.

Neurotransmission in complex animals depends on a choir of functionally distinct synapses releasing neurotransmitters in a highly coordinated manner. During synaptic signaling, vesicles fuse with the plasma membrane to release their contents. The rate of vesicle fusion is high and can exceed the rate at which synaptic vesicles can be re-supplied by distant sources. Thus, local compensatory endocytosis is needed to replenish the synaptic vesicle pools. Over the last four decades, various experimental methods and model systems have been used to study the cellular and molecular mechanisms underlying synaptic vesicle cycle. Clathrin-mediated endocytosis is thought to be the predominant mechanism for synaptic vesicle recycling. However, recent studies suggest significant contribution from other modes of endocytosis, including fast compensatory endocytosis, activity-dependent bulk endocytosis, ultrafast endocytosis, as well as kiss-and-run. Currently, it is not clear whether a universal model of vesicle recycling exist for all types of synapses. It is possible that each synapse type employs a particular mode of endocytosis. Alternatively, multiple modes of endocytosis operate at the same synapse, and the synapse toggles between different modes depending on its activity level. Here we compile review and research articles based on well-characterized model systems: frog neuromuscular junctions, C. elegans neuromuscular junctions, Drosophila neuromuscular junctions, lamprey reticulospinal giant axons, goldfish retinal ribbon synapses, the calyx of Held, and rodent hippocampal synapses. We will compare these systems in terms of their known modes and kinetics of synaptic vesicle endocytosis, as well as the underlying molecular machineries. We will also provide the future development of this field.

T-cell prolymphocytic leukemia (T-PLL), a rare type of peripheral T-cell leukemia, is characterized by marked splenomegaly with rapidly progressive lymphocytosis and a poor prognosis. Nine kinds of ABL1 chimeric genes have been identified in various kinds of hematological malignancies, such as chronic myeloid leukemia and B- or T-lymphoblastic leukemia. However, there have been no reports describing T-PLL cases with ABL1 rearrangements. We herein report a case of T-PLL with a novel SEPT9-ABL1 fusion gene which induced strong resistance to tyrosine kinase inhibitors such as imatinib and dasatinib.

A “wet chemistry” approach for isolation of 211At from an irradiated bismuth target is described. The approach involves five steps: (1) dissolution of bismuth target in conc. HNO3; (2) removal of the HNO3 by distillation; (3) dissolution of residue in 8 M HCl; (4) extraction of 211At from 8 M HCl into DIPE; and (5) extraction of 211At from DIPE into NaOH. Results from 55 “optimized” 211At isolation runs gave recovery yields of approximately 78% after decay and attenuation corrections. An attenuation-corrected average of 26 ± 3 mCi in the target provided isolated (actual) yields of 16 ± 3 mCi of 211At. A sixth step, used for purification of 211At from trace metals, was evaluated in seven runs. In those runs, isolated 211At was distilled under reductive conditions to provide an average 71 ± 8% recovery. RadioHPLC analyses of the isolated 211At solutions, both initial and after distillation, were obtained to examine the 211At species present. The primary species of 211At present was astatide, but astatate and unidentified species were also observed. Studies to determine the effect of bismuth attenuation on 211At were conducted to estimate an attenuation factor (~1.33) for adjustment of 211At readings in the bismuth target.

Electron microscopy depicts subcellular structures at synapses exquisitely but only captures static images. To visualize membrane dynamics, we have developed a novel technique, called flash-and-freeze, which induces neuronal activity with a flash of light and captures the membrane dynamics by rapid freezing. For characterizing membrane movements during synaptic transmission, a light-sensitive cation channel, channelrhodopsin, is heterologously expressed in mouse hippocampal neurons or in Caenorhabditis elegans motor neurons. A brief pulse of blue light activates channelrhodopsin and induces an action potential, leading to synaptic transmission. Following the light stimulation, neurons are frozen at different time intervals ranging from 10 ms to 20 s. Electron micrographs are then acquired from each time point to visualize the morphological changes. Using this approach, we have characterized a novel form of endocytosis, ultrafast endocytosis, which rapidly removes excess membrane added to the surface during neurotransmission. The flash-and-freeze approach can be adapted to study other cellular phenomena that can be induced by light-sensitive genetic or pharmacological tools.

Synaptic vesicles can be released at extremely high rates, which places an extraordinary demand on the recycling machinery. Previous ultrastructural studies of vesicle recycling were conducted in dissected preparations using an intense stimulation to maximize the probability of release. Here, a single light stimulus was applied to motor neurons in intact Caenorhabditis elegans nematodes expressing channelrhodopsin, and the animals rapidly frozen. We found that docked vesicles fuse along a broad active zone in response to a single stimulus, and are replenished with a time constant of about 2 s. Endocytosis occurs within 50 ms adjacent to the dense projection and after 1 s adjacent to adherens junctions. These studies suggest that synaptic vesicle endocytosis may occur on a millisecond time scale following a single physiological stimulus in the intact nervous system and is unlikely to conform to current models of endocytosis.

The clathrin adaptor complex AP2 is thought to be an obligate heterotetramer. We identify null mutations in the α subunit of AP2 in the nematode Caenorhabditis elegans. α-adaptin mutants are viable and the remaining μ2/β hemicomplex retains some function. Conversely, in μ2 mutants, the alpha/sigma2 hemicomplex is localized and is partially functional. α-μ2 double mutants disrupt both halves of the complex and are lethal. The lethality can be rescued by expression of AP2 components in the skin, which allowed us to evaluate the requirement for AP2 subunits at synapses. Mutations in either α or μ2 subunits alone reduce the number of synaptic vesicles by about 30%; however, simultaneous loss of both α and μ2 subunits leads to a 70% reduction in synaptic vesicles and the presence of large vacuoles. These data suggest that AP2 may function as two partially independent hemicomplexes.

The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Members of the stonin protein family (Drosophila Stoned B, mammalian stonin 2) have been shown to link the synaptic vesicle protein synaptotagmin to the endocytic machinery. Here we characterize the unc-41 gene, which encodes the stonin ortholog in the nematode Caenorhabditis elegans. Transgenic expression of Drosophila stonedB rescues unc-41 mutant phenotypes, demonstrating that UNC-41 is a bona fide member of the stonin family. In unc-41 mutants, synaptotagmin is present in axons, but is mislocalized and diffuse. In contrast, UNC-41 is localized normally in synaptotagmin mutants, demonstrating a unidirectional relationship for localization. The phenotype of snt-1 unc-41 double mutants is stronger than snt-1 mutants, suggesting that UNC-41 may have additional, synaptotagmin-independent functions. We also show that unc-41 mutants have defects in synaptic vesicle membrane endocytosis, including a ∼50% reduction of vesicles in both acetylcholine and GABA motor neurons. These endocytic defects are similar to those observed in apm-2 mutants, which lack the µ2 subunit of the AP2 adaptor complex. However, no further reduction in synaptic vesicles was observed in unc-41 apm-2 double mutants, suggesting that UNC-41 acts in the same endocytic pathway as µ2 adaptin.

Glial cells surround neuronal endings to create enclosed compartments required for neuronal function. This architecture is seen at excitatory synapses and at sensory neuron receptive endings. Despite the prevalence and importance of these compartments, how they form is not known. We used the main sensory organ of C. elegans, the amphid, to investigate this issue. daf-6/Patched-related is a glia-expressed gene previously implicated in amphid sensory compartment morphogenesis. By comparing time series of electron-microscopy (EM) reconstructions of wild-type and daf-6 mutant embryos, we show that daf-6 acts to restrict compartment size. From a genetic screen, we found that mutations in the gene lit-1/Nemo-like kinase (NLK) suppress daf-6. EM and genetic studies demonstrate that lit-1 acts within glia, in counterbalance to daf-6, to promote sensory compartment expansion. Although LIT-1 has been shown to regulate Wnt signaling, our genetic studies demonstrate a novel, Wnt-independent role for LIT-1 in sensory compartment size control. The LIT-1 activator MOM-4/TAK1 is also important for compartment morphogenesis and both proteins line the glial sensory compartment. LIT-1 compartment localization is important for its function and requires neuronal signals. Furthermore, the conserved LIT-1 C-terminus is necessary and sufficient for this localization. Two-hybrid and co-immunoprecipitation studies demonstrate that the LIT-1 C-terminus binds both actin and the Wiskott-Aldrich syndrome protein (WASP), an actin regulator. We use fluorescence light microscopy and fluorescence EM methodology to show that actin is highly enriched around the amphid sensory compartment. Finally, our genetic studies demonstrate that WASP is important for compartment expansion and functions in the same pathway as LIT-1. The studies presented here uncover a novel, Wnt-independent role for the conserved Nemo-like kinase LIT-1 in controlling cell morphogenesis in conjunction with the actin cytoskeleton. Our results suggest that the opposing daf-6 and lit-1 glial pathways act together to control sensory compartment size.

Synaptic vesicles dock to the plasma membrane at synapses to facilitate rapid exocytosis. Docking was originally proposed to require the soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) proteins; however, perturbation studies suggested that docking was independent of the SNARE proteins. We now find that the SNARE protein syntaxin is required for docking of all vesicles at synapses in the nematode Caenorhabditis elegans. The active zone protein UNC-13, which interacts with syntaxin, is also required for docking in the active zone. The docking defects in unc-13 mutants can be fully rescued by overexpressing a constitutively open form of syntaxin, but not by wild-type syntaxin. These experiments support a model for docking in which UNC-13 converts syntaxin from the closed to the open state, and open syntaxin acts directly in docking vesicles to the plasma membrane. These data provide a molecular basis for synaptic vesicle docking.