Control of Synaptic Levels of Nicotinic Acetylcholine Receptor by the Sequestering Subunit Dα5 and Secreted Scaffold Protein Hig.

The presentation of nicotinic acetylcholine receptors (nAChRs) on synaptic membranes is crucial for generating cholinergic circuits, some of which are associated with memory function and neurodegenerative disorders. Although the physiology and structure of nAChR, a cation channel comprising five subunits, have been extensively studied, little is known about how the receptor levels in interneuronal synapses are determined and which nAChR subunits participate in the regulatory process in cooperation with synaptic cleft matrices and intracellular proteins. By a genetic screen of Drosophila, we identified mutations in the nAChR subunit Dα5 gene as suppressors that restored the mutant phenotypes of hig, which encodes a secretory matrix protein localized to cholinergic synaptic clefts in the brain. Only the loss of function of Dα5 among the 10 nAChR subunits suppressed hig mutant phenotypes in both male and female flies. Dα5 behaved as a lethal factor when Hig was defective; loss of Dα5 in hig mutants rescued lethality, upregulating Dα6 synaptic levels. By contrast, levels of Dα5, Dα6, and Dα7 subunits were all reduced in hig mutants. These three subunits have distinct properties for interaction with Hig or trafficking, as confirmed by chimeric subunit experiments. Notably, the chimeric Dα5 protein, which has the extracellular sequences that display no positive interaction with Hig, exhibited abnormal distribution and lethality even in the presence of Hig. We propose that the sequestering subunit Dα5 functions by reducing synaptic levels of nAChR through internalization, and this process is blocked by Hig, which tethers Dα5 to the synaptic cleft matrix.SIGNIFICANCE STATEMENT Because the cholinergic synapse is one of the major synapses that generate various brain functions, numerous studies have sought to reveal the physiology and structure of the nicotinic acetylcholine receptor (nAChR). However, little is known about how synaptic levels of nAChR are controlled and which nAChR subunits participate in the regulatory process in cooperation with synaptic cleft matrices. By a genetic screen of Drosophila, we identified mutations in the nAChR subunit Dα5 gene as suppressors that restored the mutant phenotypes of hig, which encodes a secretory matrix protein localized to cholinergic synaptic clefts. Our data indicate that Dα5 functions in reducing synaptic levels of nAChR, and this process is blocked by Hig, which tethers Dα5 to the synaptic cleft matrix.

Organizing the synaptic junctions.

Synaptic adhesion molecules (SAMs) are essential for driving the formation, maturation, and plasticity of synaptic connections for neural networks. MAM domain-containing glycosylphosphatidylinositol anchors (MDGAs) are a type of SAM that regulates the formation of trans-synaptic bridges, which are critical for neurotransmission and synaptic differentiation. In a recent issue of the JBC, Lee et al. uncovered that MDGA1 can control protein-protein interactions and synaptic cleft activity by adopting different global 3D conformations. This novel molecular mechanism may be applicable to other SAMs that regulate protein-protein interactions and nanoscale organization in the synaptic cleft.

Neuronal Glutamatergic Synaptic Clefts Alkalinize Rather Than Acidify during Neurotransmission.

The dogma that the synaptic cleft acidifies during neurotransmission is based on the corelease of neurotransmitters and protons from synaptic vesicles, and is supported by direct data from sensory ribbon-type synapses. However, it is unclear whether acidification occurs at non-ribbon-type synapses. Here we used genetically encoded fluorescent pH indicators to examine cleft pH at conventional neuronal synapses. At the neuromuscular junction of female Drosophila larvae, we observed alkaline spikes of over 1 log unit during fictive locomotion in vivo. Ex vivo, single action potentials evoked alkalinizing pH transients of only ∼0.01 log unit, but these transients summated rapidly during burst firing. A chemical pH indicator targeted to the cleft corroborated these findings. Cleft pH transients were dependent on Ca2+ movement across the postsynaptic membrane, rather than neurotransmitter release per se, a result consistent with cleft alkalinization being driven by the Ca2+/H+ antiporting activity of the plasma membrane Ca2+-ATPase at the postsynaptic membrane. Targeting the pH indicators to the microenvironment of the presynaptic voltage gated Ca2+ channels revealed that alkalinization also occurred within the cleft proper at the active zone and not just within extrasynaptic regions. Application of the pH indicators at the mouse calyx of Held, a mammalian central synapse, similarly revealed cleft alkalinization during burst firing in both males and females. These findings, made at two quite different non-ribbon type synapses, suggest that cleft alkalinization during neurotransmission, rather than acidification, is a generalizable phenomenon across conventional neuronal synapses.SIGNIFICANCE STATEMENT Neurotransmission is highly sensitive to the pH of the extracellular milieu. This is readily evident in the neurological symptoms that accompany systemic acid/base imbalances. Imaging data from sensory ribbon-type synapses show that neurotransmission itself can acidify the synaptic cleft, likely due to the corelease of protons and glutamate. It is not clear whether the same phenomenon occurs at conventional neuronal synapses due to the difficulties in collecting such data. If it does occur, it would provide for an additional layer of activity-dependent modulation of neurotransmission. Our findings of alkalinization, rather than acidification, within the cleft of two different neuronal synapses encourages a reassessment of the scope of activity-dependent pH influences on neurotransmission and short-term synaptic plasticity.

Kinetics of Chemical Processes in the Human Brain. Proton Blockade of Acetylcholinesterase and pH-Impulse in the Mechanism of Functioning of the Cholinergic Synapse.

A kinetic model describing the dynamics of synaptic "discharge" taking into account the kinetics of the injection of the neurotransmitter into the synaptic cleft, the pH-dependence of the catalytic activity of the enzyme, and diffusion withdrawal of protons is proposed. The model provides a physicochemical explanation for a number of important physiological phenomena, such as the neuromuscular paralysis, the molecular mechanism of neurological memory, and the effect of some neurotoxins and drugs.

Kinetics of Chemical Processes in the Human Brain. The Cholinergic Synapse-Mechanisms of Functioning and Control Methods.

In the framework of the kinetic model, the functioning of the cholinergic synapse is considered. The results of mathematical modeling of changes in the level of acetylcholine, induced pH impulse, the influence of the frequency of impulse transmission and inhibition of acetylcholinesterase are presented. Physicochemical explanation for a number of important physiological phenomena, such as neuromuscular paralysis, the molecular mechanism of neurological memory, and actions of nerve poisons and toxins, is given.

Collagen VI is required for the structural and functional integrity of the neuromuscular junction.

The synaptic cleft of the neuromuscular junction (NMJ) consists of a highly specialized extracellular matrix (ECM) involved in synapse maturation, in the juxtaposition of pre- to post-synaptic areas, and in ensuring proper synaptic transmission. Key components of synaptic ECM, such as collagen IV, perlecan and biglycan, are binding partners of one of the most abundant ECM protein of skeletal muscle, collagen VI (ColVI), previously never linked to NMJ. Here, we demonstrate that ColVI is itself a component of this specialized ECM and that it is required for the structural and functional integrity of NMJs. In vivo, ColVI deficiency causes fragmentation of acetylcholine receptor (AChR) clusters, with abnormal expression of NMJ-enriched proteins and re-expression of fetal AChRγ subunit, both in Col6a1 null mice and in patients affected by Ullrich congenital muscular dystrophy (UCMD), the most severe form of ColVI-related myopathies. Ex vivo muscle preparations from ColVI null mice revealed altered neuromuscular transmission, with electrophysiological defects and decreased safety factor (i.e., the excess current generated in response to a nerve impulse over that required to reach the action potential threshold). Moreover, in vitro studies in differentiated C2C12 myotubes showed the ability of ColVI to induce AChR clustering and synaptic gene expression. These findings reveal a novel role for ColVI at the NMJ and point to the involvement of NMJ defects in the etiopathology of ColVI-related myopathies.

Extracorporeal shock wave treatment can selectively destroy end plates in neuromuscular junctions.

INTRODUCTION: This study assesses the effect of radial extracorporeal shock wave (rESW) exposure on neuromuscular transmission and neuromuscular junction (NMJ) morphology. METHODS: We applied 2,000 rESWs at 0.18 mJ/mm2 and a frequency of 15 Hz to the right calf of male rats, measured the compound muscle action potential (CMAP), and examined NMJ morphology using electron microscopy. Left calf muscles were used as controls. RESULTS: rESW exposure significantly reduced CMAP amplitude without delayed latency in exposed muscles compared with controls. All rESW-exposed muscles exhibited NMJs with irregular end plates. Mean interjunctional fold interval was significantly increased compared with controls. However, axon terminals and muscle fibers surrounding NMJs with irregular end plates were unchanged. DISCUSSION: This localized destruction of end plates may be caused by differences in acoustic impedance induced by the density of acetylcholine receptors. These results provide a possible mechanism for the effectiveness of rESW treatment for spasticity and dystonia. Muscle Nerve 57: 466-472, 2018.

Three-dimensional imaging of Drosophila motor synapses reveals ultrastructural organizational patterns.

We combined cryo-preservation of intact Drosophila larvae and electron tomography with comprehensive segmentation of key features to reconstruct the complete ultrastructure of a model glutamatergic synapse in a near-to-native state. Presynaptically, we detail a complex network of filaments that connects and organizes synaptic vesicles. We link the complexity of this synaptic vesicle network to proximity to the active zone cytomatrix, consistent with the model that these protein structures function together to regulate synaptic vesicle pools. We identify a net-shaped network of electron-dense filaments spanning the synaptic cleft that suggests conserved organization of trans-synaptic adhesion complexes at excitatory synapses. Postsynaptically, we characterize a regular pattern of macromolecules that yields structural insights into the scaffolding of neurotransmitter receptors. Together, these analyses reveal an unexpected level of conservation in the nanoscale organization of diverse glutamatergic synapses and provide a structural foundation for understanding the molecular machines that regulate synaptic communication at a powerful model synapse.

The matrix protein Hikaru genki localizes to cholinergic synaptic clefts and regulates postsynaptic organization in the Drosophila brain.

The synaptic cleft, a crucial space involved in neurotransmission, is filled with extracellular matrix that serves as a scaffold for synaptic differentiation. However, little is known about the proteins present in the matrix and their functions in synaptogenesis, especially in the CNS. Here, we report that Hikaru genki (Hig), a secreted protein with an Ig motif and complement control protein domains, localizes specifically to the synaptic clefts of cholinergic synapses in the Drosophila CNS. The data indicate that this specific localization is achieved by capture of secreted Hig in synaptic clefts, even when it is ectopically expressed in glia. In the absence of Hig, the cytoskeletal scaffold protein DLG accumulated abnormally in cholinergic postsynapses, and the synaptic distribution of acetylcholine receptor (AchR) subunits Dα6 and Dα7 significantly decreased. hig mutant flies consistently exhibited resistance to the AchR agonist spinosad, which causes lethality by specifically activating the Dα6 subunit, suggesting that loss of Hig compromises the cholinergic synaptic activity mediated by Dα6. These results indicate that Hig is a specific component of the synaptic cleft matrix of cholinergic synapses and regulates their postsynaptic organization in the CNS.

Role of Spirulina in Structural Remodeling of Synapse in Telencephalon of Chronic Unpredictable Stress Model of Zebrafish.

Background: Stress can affect the morphology and synaptic organization of the telencephalon. These structural changes at the cellular level can lead to the development of various psychopathologies. Purpose: Given that the telencephalon plays a major role in stress responses, the current study aimed to investigate the role of Spirulina platensis as a neuroprotectant supplement in the early life of zebrafish in averting the alteration of synapse morphology in the telencephalon caused by chronic unpredictable stress (CUS) in the later stage. Methods: 5dpf larvae were divided into two groups: one group was fed with a commercial fish diet and a second group with a 1% Spirulina-supplemented diet for 90 days. After 90 days, the adult zebrafish were exposed to CUS with different chronic stressors for 15 days. The synaptic plasticity was evaluated by morphometric analysis of synapse in telencephalon of zebrafish by transmission electron microscopy. Results: The ultrastructural study demonstrated the protective role of Spirulina in the CUS model as no significant alterations in the length of the active zone, postsynaptic density, and synaptic cleft were observed as compared to the control group in the CUS model. Conclusion: Thus, suggesting that the Spirulina supplementation can avert the remodeling effect of stress on synapse ultrastructure.

Modification of the synaptic cleft under excitatory conditions.

The synaptic cleft is the extracellular part of the synapse, bridging the pre- and postsynaptic membranes. The geometry and molecular organization of the cleft is gaining increased attention as an important determinant of synaptic efficacy. The present study by electron microscopy focuses on short-term morphological changes at the synaptic cleft under excitatory conditions. Depolarization of cultured hippocampal neurons with high K+ results in an increased frequency of synaptic profiles with clefts widened at the periphery (open clefts), typically exhibiting patches of membranes lined by postsynaptic density, but lacking associated presynaptic membranes (18.0% open clefts in high K+ compared to 1.8% in controls). Similarly, higher frequencies of open clefts were observed in adult brain upon a delay of perfusion fixation to promote excitatory/ischemic conditions. Inhibition of basal activity in cultured neurons through the application of TTX results in the disappearance of open clefts whereas application of NMDA increases their frequency (19.0% in NMDA vs. 5.3% in control and 2.6% in APV). Depletion of extracellular Ca2+ with EGTA also promotes an increase in the frequency of open clefts (16.6% in EGTA vs. 4.0% in controls), comparable to that by depolarization or NMDA, implicating dissociation of Ca2+-dependent trans-synaptic bridges. Dissociation of transsynaptic bridges under excitatory conditions may allow perisynaptic mobile elements, such as AMPA receptors to enter the cleft. In addition, peripheral opening of the cleft would facilitate neurotransmitter clearance and thus may have a homeostatic and/or protective function.

Synaptic environment and extrasynaptic glutamate signals: The quest continues.

Behaviour of a mammal relies on the brain's excitatory circuits equipped with glutamatergic synapses. In most cases, glutamate escaping from the synaptic cleft is rapidly buffered and taken up by high-affinity transporters expressed by nearby perisynaptic astroglial processes (PAPs). The spatial relationship between glutamatergic synapses and PAPs thus plays a crucial role in understanding glutamate signalling actions, yet its intricate features can only be fully appreciated using methods that operate beyond the diffraction limit of light. Here, we examine principal aspects pertaining to the receptor actions of glutamate, inside and outside the synaptic cleft in the brain, where the organisation of synaptic micro-physiology and micro-environment play a critical part. In what conditions and how far glutamate can escape the synaptic cleft activating its target receptors outside the immediate synapse has long been the subject of debate. Evidence is also emerging that neuronal activity- and astroglia-dependent glutamate spillover actions could be important across the spectrum of cognitive functions This article is part of the special issue on 'Glutamate Receptors - The Glutamatergic Synapse'.

Fine Localization of Acetylcholinesterase in the Synaptic Cleft of the Vertebrate Neuromuscular Junction.

Acetylcholinesterase (AChE) is concentrated at cholinergic synapses, where it is a major factor in controlling the duration of transmitter action. The concentration and localization of AChE within the synaptic cleft are in keeping with the functional requirements of the particular type of synapse. The densities of synaptic AChE at various neuromuscular junctions (NMJs) had been evaluated by quantitative EM-autoradiography using radiolabeled probes. Yet, fundamental issues concerning the precise distribution and location of the enzyme in the cleft remained open: whether and to what extent synaptic AChE is associated with pre- or postsynaptic membranes, or with synaptic basal lamina (BL), and whether it occurs only in the primary cleft (PC) or also in postjunctional folds (PJFs). Nanogold-conjugates of fasciculin, an anticholinesterase polypeptide toxin, were prepared and used to label AChE at NMJs of mouse and frog muscles. Selective intense labeling was obtained at the NMJs, with gold-labeled AChE sites distributed over the BL in the PC and the PJFs. Quantitative analysis demonstrated that AChE sites are almost exclusively located on the BL rather than on pre- or postsynaptic membranes and are distributed in the PC and down the PJFs, with a defined pattern. This localization pattern of AChE is suggested to ensure full hydrolysis of acetylcholine (ACh) bouncing off receptors, thus eliminating its unnecessary detrimental reattachment.

Shear-Induced Amyloid Formation in the Brain: IV. Effects on Synapses Surrounding Senile Plaque and in Plaque-Free Regions.

Amyloid-ß oligomers (AßO) have been proposed as neurotoxins in the synaptic dysfunction that precedes Alzheimer's disease symptoms. Human and animal model studies report that senile plaques contain a halo of AßO molecules surrounding these plaques. A far smaller number of oligomers are distributed widely in plaque-free regions. It has been suggested that oligomers migrate from halos to nearby synapses and are incorporated into both pre- and postsynaptic terminals. These two types of oligomers have two different toxicities when extracted and injected in animal models. This paper proposes a shear-energy based explanation for the data in these studies. Shear hypotheses in the preceding three papers in this series are applied to suggest how the hydrodynamics and resulting shear patterns explain the spatial distribution of both AßO types, the apparent synapse loss in the vicinity of plaque particles, and possible reasons for the differing toxicities. A shear-based mechanism is proposed for the preferential migration of locally shear-excited Aß molecules into the synaptic cleft. It is proposed that high energy laminar shear generated by the forced diversion of interstitial fluid around the flow-impeding plaque particle is responsible for the formation of AßOs around the plaque. It is suggested that in plaque-free regions, a different type of AßO with different toxicity is generated by lower energy shear flow around synapses, depositing AßO within the synapse from either the neuron membrane surface or by prion-like seeding within the synaptic cleft by locally-sheared Aß molecules near the synapse entry.

Modeling of Zinc Dynamics in the Synaptic Cleft: Implications for Cadherin Mediated Adhesion and Synaptic Plasticity.

While the numerous influences of synaptically released zinc on synaptic efficiency during long-term potentiation have been discussed by many authors already, we focused on the possible effect of zinc on cadherins and therefore its contribution to morphological changes in the context of synaptic plasticity. The difficulty with gaining insights into the dynamics of zinc-cadherin interaction is the inability to directly observe it on a suitable timescale. Therefore our approach was to establish an analytical model of the zinc diffusion dynamics in the synaptic cleft and experimentally validate, if the theoretical concentrations at the periphery of the synaptic cleft are sufficient to significantly modulate cadherin-mediated adhesion. Our results emphasize, that synaptically released zinc might have a strong accelerating effect on the morphological changes involved in long-term synaptic plasticity. The approach presented here might also prove useful for investigations on other synaptically released trace metals.

NMJ maintenance and repair in aging.

As the final output of the somatic nervous system, the neuromuscular junction (NMJ) is essential for all voluntary movements. The NMJ is also necessary for connected cells to function and survive. Because of this central role, much effort has been devoted to understanding the effects of aging, diseases, and injuries on the NMJ. These efforts have revealed a close relationship between aberrant changes at NMJs and its three cellular components - the presynaptic site on motor axons, the postsynaptic region on muscle fibers and perisynaptic Schwann cells. Here, we review the morphological and molecular changes associated with aging NMJs in rodents and humans. We also provide an overview of factors with potential roles in maintaining and repairing adult and aged NMJs.

Mapping the Proteome of the Synaptic Cleft through Proximity Labeling Reveals New Cleft Proteins.

Synapses are specialized neuronal cell-cell contacts that underlie network communication in the mammalian brain. Across neuronal populations and circuits, a diverse set of synapses is utilized, and they differ in their molecular composition to enable heterogenous connectivity patterns and functions. In addition to pre- and post-synaptic specializations, the synaptic cleft is now understood to be an integral compartment of synapses that contributes to their structural and functional organization. Aiming to map the cleft proteome, this study applied a peroxidase-mediated proximity labeling approach and used the excitatory synaptic cell adhesion protein SynCAM 1 fused to horseradish peroxidase (HRP) as a reporter in cultured cortical neurons. This reporter marked excitatory synapses as measured by confocal microcopy and was targeted to the edge zone of the synaptic cleft as determined using 3D dSTORM super-resolution imaging. Proximity labeling with a membrane-impermeant biotin-phenol compound restricted labeling to the cell surface, and Label-Free Quantitation (LFQ) mass spectrometry combined with ratiometric HRP tagging of membrane vs. synaptic surface proteins was used to identify the proteomic content of excitatory clefts. Novel cleft candidates were identified, and Receptor-type tyrosine-protein phosphatase zeta was selected and successfully validated. This study supports the robust applicability of peroxidase-mediated proximity labeling for synaptic cleft proteomics and its potential for understanding synapse heterogeneity in health and changes in diseases such as psychiatric disorders and addiction.

High-frequency (50 Hz) electroacupuncture ameliorates cognitive impairment in rats with amyloid beta 1-42-induced Alzheimer's disease.

Acupuncture has been shown to ameliorate cognitive impairment of Alzheimer's disease. Acupoints and stimulation frequency influence the therapeutic effect of electroacupuncture. Rat models of Alzheimer's disease were established by injecting amyloid beta 1-42 (Aß1-42) into the bilateral lateral ventricles. Electroacupuncture at 2, 30, and 50 Hz was carried out at Baihui (GV20; 15° obliquely to a depth of 2 mm) and Shenshu (BL23; perpendicularly to 4-6 mm depth), once a day for 20 minutes (each), for 15 days, taking a break every 7 days. The Morris water maze test was conducted to assess the learning and memory. The expression levels of glycogen synthase kinase-3ß (GSK-3ß), pSer9-GSK-3ß, pTyr216-GSK-3ß, amyloid precursor protein and Aß1-40 in the hippocampus were determined by western blot assay. Results demonstrated that electroacupuncture treatment at different frequencies markedly improved learning and memory ability, increased synaptic curvatures, decreased the width of synaptic clefts, thickened postsynaptic densities, and downregulated the expression of GSK-3ß, amyloid precursor protein, and Aß1-40. pSer9-GSK-3ß expression markedly decreased, while pTyr216-GSK-3ß expression increased. High-frequency (50 Hz) electroacupuncture was more effective than low (2 Hz) or medium-frequency (30 Hz) electroacupuncture. In conclusion, electroacupuncture treatment exerts a protective effect against Aß1-42-induced learning and memory deficits and synapse-ultrastructure impairment via inhibition of GSK-3ß activity. Moreover, high-frequency electroacupuncture was the most effective therapy.

Transcellular Nanoalignment of Synaptic Function.

At each of the brain's vast number of synapses, the presynaptic nerve terminal, synaptic cleft, and postsynaptic specialization form a transcellular unit to enable efficient transmission of information between neurons. While we know much about the molecular machinery within each compartment, we are only beginning to understand how these compartments are structurally registered and functionally integrated with one another. This review will describe the organization of each compartment and then discuss their alignment across pre- and postsynaptic cells at a nanometer scale. We propose that this architecture may allow for precise synaptic information exchange and may be modulated to contribute to the remarkable plasticity of brain function.