Synaptotagmin-11 Inhibits Synaptic Vesicle Endocytosis via Endophilin A1.

Synaptic vesicle (SV) endocytosis is a critical and well-regulated process for the maintenance of neurotransmission. We previously reported that synaptotagmin-11 (Syt11), an essential non-Ca2+-binding Syt associated with brain diseases, inhibits neuronal endocytosis (Wang et al., 2016). Here, we found that Syt11 deficiency caused accelerated SV endocytosis and vesicle recycling under sustained stimulation and led to the abnormal membrane partition of synaptic proteins in mouse hippocampal boutons of either sex. Furthermore, our study revealed that Syt11 has direct but Ca2+-independent binding with endophilin A1 (EndoA1), a membrane curvature sensor and endocytic protein recruiter, with high affinity. EndoA1-knockdown significantly reversed Syt11-KO phenotype, identifying EndoA1 as a main inhibitory target of Syt11 during SV endocytosis. The N-terminus of EndoA1 and the C2B domain of Syt11 were responsible for this interaction. A peptide (amino acids 314-336) derived from the Syt11 C2B efficiently blocked Syt11-EndoA1 binding both in vitro and in vivo Application of this peptide inhibited SV endocytosis in WT hippocampal neurons but not in EndoA1-knockdown neurons. Moreover, intracellular application of this peptide in mouse calyx of Held terminals of either sex effectively hampered both fast and slow SV endocytosis at physiological temperature. We thus propose that Syt11 ensures the precision of protein retrieval during SV endocytosis by inhibiting EndoA1 function at neuronal terminals.SIGNIFICANCE STATEMENT Endocytosis is a key stage of synaptic vesicle (SV) recycling. SV endocytosis retrieves vesicular membrane and protein components precisely to support sustained neurotransmission. However, the molecular mechanisms underlying the regulation of SV endocytosis remain elusive. Here, we reported that Syt11-KO accelerated SV endocytosis and impaired membrane partition of synaptic proteins. EndoA1 was identified as a main inhibitory target of Syt11 during SV endocytosis. Our study reveals a novel inhibitory mechanism of SV endocytosis in preventing hyperactivation of endocytosis, potentially safeguarding the recycling of synaptic proteins during sustained neurotransmission.

Molecular insights into the gating mechanisms of voltage-gated calcium channel CaV2.3.

High-voltage-activated R-type CaV2.3 channel plays pivotal roles in many physiological activities and is implicated in epilepsy, convulsions, and other neurodevelopmental impairments. Here, we determine the high-resolution cryo-electron microscopy (cryo-EM) structure of human CaV2.3 in complex with the α2δ1 and ß1 subunits. The VSDII is stabilized in the resting state. Electrophysiological experiments elucidate that the VSDII is not required for channel activation, whereas the other VSDs are essential for channel opening. The intracellular gate is blocked by the W-helix. A pre-W-helix adjacent to the W-helix can significantly regulate closed-state inactivation (CSI) by modulating the association and dissociation of the W-helix with the gate. Electrostatic interactions formed between the negatively charged domain on S6II, which is exclusively conserved in the CaV2 family, and nearby regions at the alpha-interacting domain (AID) and S4-S5II helix are identified. Further functional analyses indicate that these interactions are critical for the open-state inactivation (OSI) of CaV2 channels.

MC4R Deficiency Causes Dysregulation of Postsynaptic Excitatory Synaptic Transmission as a Crucial Culprit for Obesity.

Melanocortin 4 receptor (MC4R) in the paraventricular nucleus of the hypothalamus (PVH) shows bidirectional characterization in modulating food intake and energy homeostasis. We demonstrate that MC4R knockdown (KD) in the PVH can attenuate AMPA receptor (AMPAR)-mediated postsynaptic responses by altering the phosphorylation of AMPAR GluA1 subunit through the protein kinase A (PKA)-dependent signaling cascade and simultaneously lead to rapid body weight gain. Furthermore, PKA KD in the PVH engendered similar electrophysiological and behavioral phenotypes as in MC4R KD mice. Importantly, we observed that the reduction of AMPAR GluA1 expression not only led to attenuated synaptic responses but also caused body weight gain, suggesting that the aberration of synaptic responses may be one of the crucial pathogeny of obesity. Our study provides the synaptic and molecular explanations of how body weight is regulated by MC4R in the PVH.

Face image-sketch synthesis via generative adversarial fusion.

Face image-sketch synthesis is widely applied in law enforcement and digital entertainment fields. Despite the extensive progression in face image-sketch synthesis, there are few methods focusing on generating a color face image from a sketch. The existing methods pay less attention to learning the illumination or highlight distribution on the face region. However, the illumination is the key factor that makes the generated color face image looks vivid and realistic. Moreover, existing methods tend to employ some image preprocessing technologies and facial region patching approaches to generate high-quality face images, which results in the high complexity and memory consumption in practice. In this paper, we propose a novel end-to-end generative adversarial fusion model, called GAF, which fuses two U-Net generators and a discriminator by jointly learning the content and adversarial loss functions. In particular, we propose a parametric tanh activation function to learn and control illumination highlight distribution over faces, which is integrated between the two U-Net generators by an illumination distribution layer. Additionally, we fuse the attention mechanism into the second U-Net generator of GAF to keep the identity consistency and refine the generated facial details. The qualitative and quantitative experiments on the public benchmark datasets show that the proposed GAF has better performance than existing image-sketch synthesis methods in synthesized face image quality (FSIM) and face recognition accuracy (NLDA). Meanwhile, the good generalization ability of GAF has also been verified. To further demonstrate the reliability and authenticity of face images generated using GAF, we use the generated face image to attack the well-known face recognition system. The result shows that the face images generated by GAF can maintain identity consistency and well maintain everyone's unique facial characteristics, which can be further used in the benchmark of facial spoofing. Moreover, the experiments are implemented to verify the effectiveness and rationality of the proposed parametric tanh activation function and attention mechanism in GAF.

Structural basis for modulation of human NaV1.3 by clinical drug and selective antagonist.

Voltage-gated sodium (NaV) channels play fundamental roles in initiating and propagating action potentials. NaV1.3 is involved in numerous physiological processes including neuronal development, hormone secretion and pain perception. Here we report structures of human NaV1.3/ß1/ß2 in complex with clinically-used drug bulleyaconitine A and selective antagonist ICA121431. Bulleyaconitine A is located around domain I-II fenestration, providing the detailed view of the site-2 neurotoxin binding site. It partially blocks ion path and expands the pore-lining helices, elucidating how the bulleyaconitine A reduces peak amplitude but improves channel open probability. In contrast, ICA121431 preferentially binds to activated domain IV voltage-sensor, consequently strengthens the Ile-Phe-Met motif binding to its receptor site, stabilizes the channel in inactivated state, revealing an allosterically inhibitory mechanism of NaV channels. Our results provide structural details of distinct small-molecular modulators binding sites, elucidate molecular mechanisms of their action on NaV channels and pave a way for subtype-selective therapeutic development.

Identification and characterization of long non-coding RNA Carip in modulating spatial learning and memory.

CaMKII has long been known to be a key effector for synaptic plasticity. Recent studies have shown that a variety of modulators interact with the subunits of CaMKII to regulate the long-term potentiation (LTP) of hippocampal neurons. However, whether long non-coding RNAs modulate the activity of CaMKII and affect synaptic plasticity is still elusive. Here, we identify a previously uncharacterized long non-coding RNA Carip that functions as a scaffold, specifically interacts with CaMKIIß, and regulates the phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartate (NMDA) receptor subunits in the hippocampus. The absence of Carip causes dysfunction of synaptic transmission and attenuates LTP in hippocampal CA3-CA1 synapses, which further leads to impairment of spatial learning and memory. In summary, our findings demonstrate that Carip modulates long-term synaptic plasticity by changing AMPA receptor and NMDA receptor activities, thereby affecting spatial learning and memory in mice.

Random Shapley Forests: Cooperative Game-Based Random Forests With Consistency.

The original random forests (RFs) algorithm has been widely used and has achieved excellent performance for the classification and regression tasks. However, the research on the theory of RFs lags far behind its applications. In this article, to narrow the gap between the applications and the theory of RFs, we propose a new RFs algorithm, called random Shapley forests (RSFs), based on the Shapley value. The Shapley value is one of the well-known solutions in the cooperative game, which can fairly assess the power of each player in a game. In the construction of RSFs, RSFs use the Shapley value to evaluate the importance of each feature at each tree node by computing the dependency among the possible feature coalitions. In particular, inspired by the existing consistency theory, we have proved the consistency of the proposed RFs algorithm. Moreover, to verify the effectiveness of the proposed algorithm, experiments on eight UCI benchmark datasets and four real-world datasets have been conducted. The results show that RSFs perform better than or at least comparable with the existing consistent RFs, the original RFs, and a classic classifier, support vector machines.

Intestinal microbiota modulates adrenomedullary response through Nod1 sensing in chromaffin cells.

The intestinal microbiota closely interacts with the neuroendocrine system and exerts profound effects on host physiology. Here, we report that nucleotide-binding oligomerization domain 1 (Nod1) ligand derived from intestinal bacteria modulates catecholamine storage and secretion in mouse adrenal chromaffin cells. The cytosolic peptidoglycan receptor Nod1 is involved in chromogranin A (Chga) retention in dense core granules (DCGs) in chromaffin cells. Mechanistically, upon recognizing its ligand, Nod1 localizes to DCGs, and recruits Rab2a, which is critical for Chga and epinephrine retention in DCGs. Depletion of Nod1 ligand or deficiency of Nod1 leads to a profound defect in epinephrine storage in chromaffin cells and subsequently less secretion upon stimulation. The intestine-adrenal medulla cross talk bridged by Nod1 ligand modulates adrenal medullary responses during the immobilization-induced stress response in mice. Thus, our study uncovers a mechanism by which intestinal microbes modulate epinephrine secretion in response to stress, which may provide further understanding of the gut-brain axis.

Measurement of intact quantal packet of transmitters released from single nerve terminal by loose-patch amperometry.

Neuronal information is majorly encoded chemically at synapses and the elementary unit of synaptic transmission is the contents of neurotransmitter released from single vesicle. However, the contents of quantal neurotransmitter have never been precisely estimated at synapses, which largely prevent our understanding the nature of quantal neurotransmitter release and its impact on neuronal information processing. In order to break through the technical bottleneck of precisely counting quantal neurotransmitter molecules, we developed a new approach in combination of electrophysiology and electrochemistry to measure intact quantal content of single vesicles. An etched submicro-carbon fiber electrode for electrochemical detection was designed to be enclosed in an electrophysiologically used glass pipette. The glass pipette allowed the electrochemical electrode to access the release site, and amperometric recordings were made within the enclosed space at the electrophysiological loose-patch mode. Our study showed that the intact quantal release could be successfully detected at the dopaminergic varicosities by this loose-patch amperometric measurement in real time with negligible leakage.

A comprehensive study on the combustion kinetic modeling of typical electronic plastic waste-television set (TV) plastic shell.

Electronic waste is the fastest growing waste stream and one of the most significant constituents is electronic plastics. In this study, the combustion kinetic of typical electronic plastic waste-television set (TV) plastic shell-was investigated using two basic kinetic methods. The reaction mechanism and kinetic compensation effect were probed as well. The thermogravimetric analysis (TGA) revealed that its degradation process can be divided into four stages, namely, reaction initiation stage (20-300 °C), major reaction stage (300-450 °C), minor reaction stage (450-600 °C), and reaction cessation stage (600-1,000 °C). The activation energy (E) were calculated and indicated that, the kinetic parameters from six model-free methods gradually decreased with α increasing from 0.1 to 0.35, and then slightly increased. The Flynn--Wall--Ozawa (FWO) method was more reliable and E values decreased from 155.0 to 147.51 kJ/mol with α range of 0.1-0.35, then gradually increased to 165.21 kJ/mol. Within the Coats--Redfern method, the first-order (F1) model had higher coefficient of determination (R2) and comparable E values with that from FWO method. The result of kinetic compensation effect confirmed that the compensation effect existed between E and A during the plastic waste combustion. A linear relationship lnA = 0.183E-3.11 (R2 = 0.991) was obtained. The pre-exponential factors (A) were also determined as 7.67 × 1010 min-1 based on the F1 reaction model and FWO method.Implications: Municipal solid waste (MSW) is a complex mixture of different components and the plastic takes up a significant portion in total MSW. Understanding the combustion process of typical electronic plastic waste and further probing its combustion kinetic are significant. Through this study, it will be significant for the reactor designing and optimizing in practice.

Synapse and Active Zone Assembly in the Absence of Presynaptic Ca2+ Channels and Ca2+ Entry.

Presynaptic CaV2 channels are essential for Ca2+-triggered exocytosis. In addition, there are two competing models for their roles in synapse structure. First, Ca2+ channels or Ca2+ entry may control synapse assembly. Second, active zone proteins may scaffold CaV2s to presynaptic release sites, and synapse structure is CaV2 independent. Here, we ablated all three CaV2s using conditional knockout in cultured hippocampal neurons or at the calyx of Held, which abolished evoked exocytosis. Compellingly, synapse and active zone structure, vesicle docking, and transsynaptic nano-organization were unimpaired. Similarly, long-term blockade of action potentials and Ca2+ entry did not disrupt active zone assembly. Although CaV2 knockout impaired the localization of ß subunits, α2δ-1 localized normally. Rescue with CaV2 restored exocytosis, and CaV2 active zone targeting depended on the intracellular C-terminus. We conclude that synapse assembly is independent of CaV2s or Ca2+ entry through them. Instead, active zone proteins recruit and anchor CaV2s via CaV2 C-termini.

Banzhaf random forests: Cooperative game theory based random forests with consistency.

Random forests algorithms have been widely used in many classification and regression applications. However, the theory of random forests lags far behind their applications. In this paper, we propose a novel random forests classification algorithm based on cooperative game theory. The Banzhaf power index is employed to evaluate the power of each feature by traversing possible feature coalitions. Hence, we call the proposed algorithm Banzhaf random forests (BRFs). Unlike the previously used information gain ratio, which only measures the power of each feature for classification and pays less attention to the intrinsic structure of the feature variables, the Banzhaf power index can measure the importance of each feature by computing the dependency among the group of features. More importantly, we have proved the consistency of BRFs, which narrows the gap between the theory and applications of random forests. Extensive experiments on several UCI benchmark data sets and three real world applications show that BRFs perform significantly better than existing consistent random forests on classification accuracy, and better than or at least comparable with Breiman's random forests, support vector machines (SVMs) and k-nearest neighbors (KNNs) classifiers.

Sr2+ has low efficiency in regulating spontaneous release at the Calyx of Held synapses.

It has been known that Ca2+ plays an essential role in mediating different modes of neurotransmitter release via different sensing mechanisms. Synaptotagmin 1, 2, and 9 were found to act as the Ca2+ sensors for synchronous release and synaptotagmin 7 and Doc-2 were proposed as the Ca2+ sensors for asynchronous release. Comparatively, the Ca2+ sensor for spontaneous release remains a mystery. At the Calyx of Held synapse, the Ca2+ sensor for spontaneous release was found not identical to the sensor for synchronous release, synaptotagmin 2. As Ca2+ sensors have different sensitivity to Sr2+ and Ca2+ and induce significantly different rate of vesicle release, Sr2+ is traditionally used as a tool to examine the intrinsic properties of different Ca2+ sensors. Here, we employed cell-attached patch recording and presynaptic/postsynaptic whole-cell recording at the Calyx of Held synapses of synaptotagmin 2 knock-out mice to assay the Sr2+ and Ca2+ influx into the nerve terminal at resting potential and observed the effects of Ca2+ and Sr2+ on spontaneous neurotransmitter release. We found that the dwell time of single voltage gated Ca2+ channel opening increased around threefold for Sr2+ than Ca2+ with the channel conductance unchanged; the divalent cation sensing machinery in regulating spontaneous release has much lower sensitivity to Sr2+ than Ca2+ . Thus, our study reveals some of the intrinsic properties of Ca2+ sensor(s) of spontaneous transmitter release and provided an insight into the underlying mechanisms.

Ion- and water-binding sites inside an occluded hourglass pore of a trimeric intracellular cation (TRIC) channel.

BACKGROUND: Trimeric intracellular cation (TRIC) channels are crucial for Ca2+ handling in eukaryotes and are involved in K+ uptake in prokaryotes. Recent studies on the representative members of eukaryotic and prokaryotic TRIC channels demonstrated that they form homotrimeric units with the ion-conducting pores contained within each individual monomer. RESULTS: Here we report detailed insights into the ion- and water-binding sites inside the pore of a TRIC channel from Sulfolobus solfataricus (SsTRIC). Like the mammalian TRIC channels, SsTRIC is permeable to both K+ and Na+ with a slight preference for K+, and is nearly impermeable to Ca2+, Mg2+, or Cl-. In the 2.2-Å resolution K+-bound structure of SsTRIC, ion/water densities have been well resolved inside the pore. At the central region, a filter-like structure is shaped by the kinks on the second and fifth transmembrane helices and two nearby phenylalanine residues. Below the filter, the cytoplasmic vestibule is occluded by a plug-like motif attached to an array of pore-lining charged residues. CONCLUSIONS: The asymmetric filter-like structure at the pore center of SsTRIC might serve as the basis for the channel to bind and select monovalent cations. A Velcro-like plug-pore interacting model has been proposed and suggests a unified framework accounting for the gating mechanisms of prokaryotic and eukaryotic TRIC channels.

Spontaneous Vesicle Release Is Not Tightly Coupled to Voltage-Gated Calcium Channel-Mediated Ca2+ Influx and Is Triggered by a Ca2+ Sensor Other Than Synaptotagmin-2 at the Juvenile Mice Calyx of Held Synapses.

It is well known that voltage-gated calcium channels (VGCCs)-mediated Ca(2+) influx triggers evoked synaptic vesicle release. However, the mechanisms of Ca(2+) regulation of spontaneous miniature vesicle release (mini) remain poorly understood. Here we show that blocking VGCCs at the juvenile mice (C57BL/6) calyx of Held synapse failed to cause an immediate change in minis. Instead, it resulted in a significant reduction (∼40%) of mini frequency several minutes after the blockage. By recording VGCC activity and single vesicle fusion events directly at the presynaptic terminal, we found that minis did not couple to VGCC-mediated Ca(2+) entry, arguing for a lack of direct correlation between mini and transient Ca(2+) influx. Moreover, mini frequencies displayed a lower apparent Ca(2+) cooperativity than those of evoked release. In agreement with this observation, abrogation of the Ca(2+) sensor synaptotagmin-2 had no effect on apparent Ca(2+) cooperativity of minis. Together, our study provides the first direct evidence that spontaneous minis are not mediated by transient Ca(2+) signals through VGCCs and are triggered by a Ca(2+)-sensing mechanism that is different from the evoked release at these microdomain VGCC-vesicle coupled synapses.

Quantitative analysis of vesicle recycling at the calyx of Held synapse.

Vesicle recycling is pivotal for maintaining reliable synaptic signaling, but its basic properties remain poorly understood. Here, we developed an approach to quantitatively analyze the kinetics of vesicle recycling with exquisite signal and temporal resolution at the calyx of Held synapse. The combination of this electrophysiological approach with electron microscopy revealed that ∼80% of vesicles (∼270,000 out of ∼330,000) in the nerve terminal are involved in recycling. Under sustained stimulation, recycled vesicles start to be reused in tens of seconds when ∼47% of the preserved vesicles in the recycling pool (RP) are depleted. The heterogeneity of vesicle recycling as well as two kinetic components of RP depletion revealed the existence of a replenishable pool of vesicles before the priming stage and led to a realistic kinetic model that assesses the size of the subpools of the RP. Thus, our study quantified the kinetics of vesicle recycling and kinetically dissected the whole vesicle pool in the calyceal terminal into the readily releasable pool (∼0.6%), the readily priming pool (∼46%), the premature pool (∼33%), and the resting pool (∼20%).

Microsecond dissection of neurotransmitter release: SNARE-complex assembly dictates speed and Ca²âº sensitivity.

SNARE-complex assembly mediates synaptic vesicle fusion during neurotransmitter release and requires that the target-SNARE protein syntaxin-1 switches from a closed to an open conformation. Although many SNARE proteins are available per vesicle, only one to three SNARE complexes are minimally needed for a fusion reaction. Here, we use high-resolution measurements of synaptic transmission in the calyx-of-Held synapse from mutant mice in which syntaxin-1 is rendered constitutively open and SNARE-complex assembly is enhanced to examine the relation between SNARE-complex assembly and neurotransmitter release. We show that enhancing SNARE-complex assembly dramatically increases the speed of evoked release, potentiates the Ca(2+)-affinity of release, and accelerates fusion-pore expansion during individual vesicle fusion events. Our data indicate that the number of assembled SNARE complexes per vesicle during fusion determines the presynaptic release probability and fusion kinetics and suggest a mechanism whereby proteins (Munc13 or RIM) may control presynaptic plasticity by regulating SNARE-complex assembly.

Bulk endocytosis at neuronal synapses.

Neurotransmitter-containing synaptic vesicle (SV) fusion with the nerve terminal plasma membrane initiates neurotransmission in response to neuronal excitation. Under mild stimulation, the fused vesicular membrane is retrieved via kiss-and-run and/or clathrin-mediated endocytosis, which is sufficient to maintain recycling of SVs. When neurons are challenged with very high stimulation, the number of fused SVs can be extremely high, resulting in significant plasma membrane addition. Under such conditions, a higher capacity retrieval pathway, bulk endocytosis, is activated to redress this large membrane imbalance. Despite first being described more than 40 years ago, the molecular mechanisms underpinning this important process have yet to be clearly defined. In this review, we highlight the current evidence for bulk endocytosis and its prevalence in various neuronal models, as well as discuss the underlying molecular components.

Emotional regulation of pain: the role of noradrenaline in the amygdala.

The perception of pain involves the activation of the spinal pathway as well as the supra-spinal pathway, which targets brain regions involved in affective and cognitive processes. Pain and emotions have the capacity to influence each other reciprocally; negative emotions, such as depression and anxiety, increase the risk for chronic pain, which may lead to anxiety and depression. The amygdala is a key-player in the expression of emotions, receives direct nociceptive information from the parabrachial nucleus, and is densely innervated by noradrenergic brain centers. In recent years, the amygdala has attracted increasing interest for its role in pain perception and modulation. In this review, we will give a short overview of structures involved in the pain pathway, zoom in to afferent and efferent connections to and from the amygdala, with emphasis on the direct parabrachio-amygdaloid pathway and discuss the evidence for amygdala's role in pain processing and modulation. In addition to the involvement of the amygdala in negative emotions during the perception of pain, this brain structure is also a target site for many neuromodulators to regulate the perception of pain. We will end this article with a short review on the effects of noradrenaline and its role in hypoalgesia and analgesia.