Distortion of homeostatic signaling proteins by simulated microgravity in rat hypothalamus: A16O/18O‐labeled comparative integrated proteomic approach

Microgravity generates oxidative stress in central nervous system, causing distortion of various vital signaling cascades involved in many homeostatic functions. Here, we performed comparative ¹⁶O/¹⁸O labeled integrated proteomic strategy to observe the differential expression of signaling proteins involved in homeostasis. In this study, rat‐tail suspension model is employed to induce simulated microgravity in CNS. By wide proteomic analysis, total of 35 and 97 significantly differentially expressed proteins were found by HPLC/ESI‐TOF and HPLC‐Q‐TOF analysis, respectively. Among the total of 132 proteins quantified, 25 proteins were found related to various signaling cascades. Protein Thy‐1, 14‐3‐3 gamma, 14‐3‐3 epsilon, 14‐3‐3 theta, 14‐3‐3 eta, and 14‐3‐3 beta/alpha proteins, calmodulin and calcium/calmodulin‐dependent protein kinase type‐II subunit beta were found upregulated under the influence of simulated microgravity. These proteins are found involved in disrupting homeostatic pathways like sleep/wake cycle, drinking behavior, hypothalamic‐pituitary‐adrenocortical regulation and fight and/or flee actions under stress. Furthermore, MS results for protein Thy‐1 were verified by Western blot analysis showing the quantification accuracy of MS instruments. Results presented here will serve as means to understand the mechanism of action of microgravity and further reference for future detailed study of consequences of microgravity on astronauts and their possible countermeasures.     

Differential expression of specific cellular defense proteins in rat hypothalamus under simulated microgravity induced conditions: Comparative proteomics

Microgravity severely halts the structural and functional cerebral capacity of astronauts especially affecting their brains due to the stress produced by cephalic fluid shift. We employed a rat tail suspension model to substantiate simulated microgravity (SM) in brain. In this study, comparative mass spectrometry was applied in order to demonstrate the differential expression of 17 specific cellular defense proteins. Gamma‐enolase, peptidyl‐prolyl cis‐trans isomerase A, glial fibrillary acidic protein, heat shock protein HSP 90‐alpha, 10 kDa heat shock protein, mitochondrial, heat shock cognate 71 kDa protein, superoxide dismutase 1 and dihydropyrimidinase‐related protein 2 were found to be upregulated by HPLC/ESI‐TOF. Furthermore, five differentially expressed proteins including 60 kDa heat shock protein, mitochondrial, heat shock protein HSP 90‐beta, peroxiredoxin‐2, stress‐induced‐phosphoprotein, and UCHL‐1 were found to be upregulated by HPLC/ESI‐Q‐TOF MS. In addition, downregulated proteins include cytochrome C, superoxide dismutase 2, somatic, and excitatory amino acid transporter 1 and protein DJ‐1. Validity of MS results was successfully performed by Western blot analysis of DJ‐1 protein. This study will not only help to understand the neurochemical responses produced under microgravity but also will give future direction to cure the proteomic losses and their after effects in astronauts.     

Glutamic Acid and γ-Aminobutyric Acid Modulate Each Other's Release Through Heterocarriers Sited on the Axon Terminals of Rat Brain

Abstract: The effects of γ-aminobutyric acid (GABA) on the spontaneous release of endogenous glutamic acid (Glu) or aspartic acid (Asp) and the effects of Glu on the release of endogenous GABA or [³H]GABA were studied in superfused rat cerebral cortex synaptosomes. GABA increased the outflow of Glu (EC₅₀17.2 μM) and Asp (EC₅₀ 18.4 μM). GABA was not antagonized by bicuculline or picrotoxin. Neither muscimol nor (-)-baclofen mimicked GABA. The effects of GABA were prevented by GABA uptake inhibitors and were Na⁺ dependent. Glu enhanced the release of [³H]GABA (EC₅₀ 11.5 μM) from cortical synaptosomes. Glu was not mimicked by the glutamate receptor agonists N-methyl-d -aspartic, kainic, or quisqualic acid. The Glu effect was decreased by the Glu uptake inhibitor D-threo-hydroxyaspartic acid (THA) and it was Na⁺ sensitive. Similarly to Glu, D-Asp increased [³H]GABA release (EC₅₀ 9.9 μM), an effect blocked by THA. Glu also increased the release of endogenous GABA from cortex synaptosomes. In this case the effect was in part blocked by the (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist 6-cyano-7-nitroquinoxaiine-2, 3-dione, whereas the 6-cyano-7-nitroquinoxaline- 2, 3-dione-insensitive portion of the effect was prevented by THA. GABA increased the [³H]D-Asp outflow (EC₅₀ 13.7 μM) from hippocampal synaptosomes in a muscimol-, (-)- baclofen-, bicuculline-, and picrotoxin-insensitive manner. The GABA effect was abolished by blocking GABA uptake and was Na⁺ dependent. Glu increased the release of [³H]- GABA from hippocampal synaptosomes (EC₅₀ 7.1 μM) in an N-methyl-d -aspartic acid-, kainic acid-, or quisqualic acid-insensitive way. The effect of Glu was prevented by THA and was Na⁺ dependent. As in the cortex, the effect of Glu was mimicked by D-Asp in a THA-sensitive manner. It is proposed that high-affinity GABA or Glu heterocarriers are sited respectively on glutamatergic or GA- BAergic nerve terminals in rat cerebral cortex and hippocampus. The uptake of GABA may modulate Glu and Asp release, whereas the uptake of Glu may modulate the release of GABA. The existence of these heterocarriers is in keeping with the reported colocalization of GABA and Glu in some cortical and hippocampal neurons. Preliminary data suggest that these mechanisms may also be present in rat cerebellum and spinal cord.     

Differential expression of synaptic proteins in unilateral 6‐OHDA lesioned rat model—A comparative proteomics approach

Parkinson's disease (PD) is characterized as a movement disorder due to lesions in the basal ganglia. As the major input region of the basal ganglia, striatum plays a vital role in coordinating movements. It receives afferents from the cerebral cortex and projects afferents to the internal segment of the globus pallidus and substantia nigra pars reticulate. Additionally, accumulating evidences support a role for synaptic dysfunction in PD. Therefore, the present study explores the changes in protein abundance involved in synaptic disorders in unilateral lesioned 6‐OHDA rat model. Based on ¹⁸O/¹⁶O‐labeling technique, striatal proteins were separated using online 2D‐LC, and identified by nano‐ESI‐quadrupole‐TOF. A total of 370 proteins were identified, including 76 significantly differentially expressed proteins. Twenty‐two downregulated proteins were found in composition of vesicle, ten of which were involved in neuronal transmission and recycling across synapses. These include N‐ethylmaleimide‐sensitive fusion protein attachment receptor proteins (SNAP‐25, syntaxin‐1A, syntaxin‐1B, VAMP2), synapsin‐1, septin‐5, clathrin heavy chain 1, AP‐2 complex subunit beta, dynamin‐1, and endophilin‐A1. Moreover, MS result for syntaxin‐1A was confirmed by Western blot analysis. Overall, these synaptic changes induced by neurotoxin may serve as a reference for understanding the functional mechanism of striatum in PD.     

SM蛋白在膜泡运输中的功能

大多数细胞内都包含靶向不同细胞器的各种运输囊泡,其运输机制在进化上是高度保守的。Sec1/Munc-18(SM)蛋白在膜泡运输中起着重要的调控作用,它能够与SNARE(Soluble N-ethylmaleimide-sensitive factorattachment protein receptor)蛋白结合,共同在细胞内各个膜融合发生部位发挥重要作用。SM蛋白和SNARE复合体中的Syntaxin蛋白结合,调节SNARE复合体的装配,并与SNARE协同作用促进整个膜融合过程。文章对SM蛋白在结构和功能分析方面的最新研究进展进行了概述。     

The Sec1/Munc18 protein, Vps33p, functions at the endosome and the vacuole of Saccharomyces cerevisiae

The Sec1/Munc18 (SM) family of proteins is thought to impart compartmental specificity to vesicle fusion reactions. Here we report characterization of Vps33p, an SM family member previously thought to act exclusively at the vacuolar membrane with the vacuolar syntaxin Vam3p. Vacuolar morphology of vps33Delta cells resembles that of cells lacking both Vam3p and the endosomal syntaxin Pep12p, suggesting that Vps33p may function with these syntaxins at the vacuole and the endosome. Consistent with this, vps33 mutants secrete the Golgi precursor form of the vacuolar hydrolase CPY into the medium. We also demonstrate that Vps33p acts at other steps, for vps33 mutants show severe defects in endocytosis at the late endosome. At the endosome, Vps33p and other class C members exist as a complex with Vps8p, a protein previously known to act in transport between the late Golgi and the endosome. Vps33p also interacts with Pep12p, a known interactor of the SM protein Vps45p. High copy PEP7/VAC1 suppresses vacuolar morphology defects of vps33 mutants. These findings demonstrate that Vps33p functions at multiple trafficking steps and is not limited to action at the vacuolar membrane. This is the first report demonstrating the involvement of a single syntaxin with two SM proteins at the same organelle.     

The Proteins Synaptotagmin and Syntaxin Are Not General Targets of Lambert-Eaton Myasthenic Syndrome Autoantibody

Abstract: Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune neuromuscular disease in which impairment of Ca²⁺ entry into the nerve ending and consequent impaired release of acetylcholine (ACh) results in muscle weakness. The identity of the primary antigenic target molecule(s) of the autoantibodies is uncertain. Electrophysiological studies and ⁴⁵Ca²⁺ uptake studies implicate a direct effect on the Ca²⁺ channel complex at the motor nerve terminal. Some recent studies, however, suggest a more indirect interference caused by binding of autoantibodies to synaptotagmin or syntaxin, molecules presumed to be involved in docking and/or coupling the synaptic vesicles to the Ca²⁺ channels in the active zone for vesicle exocytosis and transmitter release. Western blot analyses of rat and human brain membrane proteins and pure recombinant synaptotagmin and syntaxin were used to examine directly the targets of LEMS autoantibodies and determine specifically whether or not synaptotagmin and/or syntaxin were general targets in LEMS. IgG from 14 patients with LEMS was used to probe western blots of gels containing synaptotagmin, syntaxin, rat synaptosomal proteins, and human brain membrane proteins. Several similar immunoreactive bands were observed using both rat and human brain membranes. These include high-molecular-weight protein bands whose size would be consistent with being components of Ca²⁺ channels. No reactive component was observed against either syntaxin or synaptotagmin in IgG of the 14 LEMS patients. However, both human and rat brain membranes contain proteins recognized by antibodies directed against synaptotagmin or syntaxin, indicating their immunologic relatedness and evolutionary conservation. These results suggest that large-molecular-weight proteins consistent with being Ca²⁺ channel subunits rather than syntaxin and synaptotagmin are general targets of LEMS autoantibodies.     

Three-dimensional structure of the rSly1 N-terminal domain reveals a conformational change induced by binding to syntaxin 5

Sec1/Mun18-like (SM) proteins and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play central roles in intracellular membrane fusion. Diverse modes of interaction between SM proteins and SNAREs from the syntaxin family have been described. However, the observation that the N-terminal domains of Sly1 and Vps45, the SM proteins involved in traffic at the endoplasmic reticulum, the Golgi, the trans-Golgi network and the endosomes, bind to similar N-terminal sequences of their cognate syntaxins suggested a unifying theme for SM protein/SNARE interactions in most internal membrane compartments. To further understand this mechanism of SM protein/SNARE coupling, we have elucidated the structure in solution of the isolated N-terminal domain of rat Sly1 (rSly1N) and analyzed its complex with an N-terminal peptide of rat syntaxin 5 by NMR spectroscopy. Comparison with the crystal structure of a complex between Sly1p and Sed5p, their yeast homologues, shows that syntaxin 5 binding requires a striking conformational change involving a two-residue shift in the register of the C-terminal beta-strand of rSly1N. This conformational change is likely to induce a significant alteration in the overall shape of full-length rSly1 and may be critical for its function. Sequence analyses indicate that this conformational change is conserved in the Sly1 family but not in other SM proteins, and that the four families represented by the four SM proteins found in yeast (Sec1p, Sly1p, Vps45p and Vps33p) diverged early in evolution. These results suggest that there are marked distinctions between the mechanisms of action of each of the four families of SM proteins, which may have arisen from different regulatory requirements of traffic in their corresponding membrane compartments.     

Function of SM protein in vesicle transport

Most cells contain various transport vesicles that target to different destinations. The underlying molecular mechanisms are highly conserved in evolution. Sec1/Munc-18 (SM) proteins play an important role on regulating vesicle transport by interacting with soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) at each vesicle fusion sites. SM proteins interact with syntaxin, an important component in SNARE complex, to regulate the assembly of SNARE complex, and promote overall membrane fusion process together with SNARE complex. This review summaries new research progresses of structure and function of SM protein.     

A Targeted siRNA Screen to Identify SNAREs Required for Constitutive Secretion in Mammalian Cells

The role of SNAREs in mammalian constitutive secretion remains poorly defined. To address this, we have developed a novel flow cytometry‐based assay for measuring constitutive secretion and have performed a targeted SNARE and Sec1/Munc18 (SM) protein‐specific siRNA screen (38 SNAREs, 4 SNARE‐like proteins and 7 SM proteins). We have identified the endoplasmic reticulum (ER)/Golgi SNAREs syntaxin 5, syntaxin 17, syntaxin 18, GS27, SLT1, Sec20, Sec22b, Ykt6 and the SM protein Sly1, along with the post‐Golgi SNAREs SNAP‐29 and syntaxin 19, as being required for constitutive secretion. Depletion of SNAP‐29 or syntaxin 19 causes a decrease in the number of fusion events at the cell surface and in SNAP‐29‐depleted cells causes an increase in the number of docked vesicles at the plasma membrane as determined by total internal reflection fluorescence (TIRF) microscopy. Analysis of syntaxin 19‐interacting partners by mass spectrometry indicates that syntaxin 19 can form SNARE complexes with SNAP‐23, SNAP‐25, SNAP‐29, VAMP3 and VAMP8, supporting its role in Golgi to plasma membrane transport or fusion. Surprisingly, we have failed to detect any requirement for a post‐Golgi‐specific R‐SNARE in this process.     

Mechanism of neurotransmitter release coming into focus

Research for three decades and major recent advances have provided crucial insights into how neurotransmitters are released by Ca²⁺‐triggered synaptic vesicle exocytosis, leading to reconstitution of basic steps that underlie Ca²⁺‐dependent membrane fusion and yielding a model that assigns defined functions for central components of the release machinery. The soluble N‐ethyl maleimide sensitive factor attachment protein receptors (SNAREs) syntaxin‐1, SNAP‐25, and synaptobrevin‐2 form a tight SNARE complex that brings the vesicle and plasma membranes together and is key for membrane fusion. N‐ethyl maleimide sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs) disassemble the SNARE complex to recycle the SNAREs for another round of fusion. Munc18‐1 and Munc13‐1 orchestrate SNARE complex formation in an NSF‐SNAP‐resistant manner by a mechanism whereby Munc18‐1 binds to synaptobrevin and to a self‐inhibited “closed” conformation of syntaxin‐1, thus forming a template to assemble the SNARE complex, and Munc13‐1 facilitates assembly by bridging the vesicle and plasma membranes and catalyzing opening of syntaxin‐1. Synaptotagmin‐1 functions as the major Ca²⁺ sensor that triggers release by binding to membrane phospholipids and to the SNAREs, in a tight interplay with complexins that accelerates membrane fusion. Many of these proteins act as both inhibitors and activators of exocytosis, which is critical for the exquisite regulation of neurotransmitter release. It is still unclear how the actions of these various proteins and multiple other components that control release are integrated and, in particular, how they induce membrane fusion, but it can be expected that these fundamental questions can be answered in the near future, building on the extensive knowledge already available.     

Characterization of VAMP‐2 in the lung: implication in lung surfactant secretion

Lung surfactant is crucial for reducing the surface tension of alveolar space, thus preventing the alveoli from collapse. Lung surfactant is synthesized in alveolar epithelial type II cells and stored in lamellar bodies before being released via the fusion of lamellar bodies with the apical plasma membrane. SNAREs (soluble N‐ethylmaleimide‐sensitive fusion protein‐attachment protein receptors) play an essential role in membrane fusion. We have previously demonstrated the requirement of t‐SNARE (target SNARE) proteins, syntaxin 2 and SNAP‐23 (N‐ethylmaleimide‐sensitive factor‐attachment protein 23), in regulated surfactant secretion. Here, we characterized the distribution of VAMPs (vesicle‐associated membrane proteins) in rat lung and alveolar type II cells. VAMP‐2, ?3 and ?8 are shown in type II cells at both mRNA and protein levels. VAMP‐2 and ?8 were enriched in LB (lamellar body) fraction. Immunochemistry studies indicated that VAMP‐2 was co‐localized with the LB marker protein, LB‐180. Functionally, the cytoplasmic domain of VAMP‐2, but not VAMP‐8 inhibited surfactant secretion in type II cells. We suggest that VAMP‐2 is the v‐SNARE (vesicle SNARE) involved in regulated surfactant secretion.     

Modification of growth anisotropy and cortical microtubule dynamics in Arabidopsis hypocotyls grown under microgravity conditions in space

We carried out a space experiment, denoted as Aniso Tubule, to examine the effects of microgravity on the growth anisotropy and cortical microtubule dynamics in Arabidopsis hypocotyls, using lines in which microtubules are visualized by labeling tubulin or microtubule‐associated proteins (MAPs) with green fluorescent protein (GFP). In all lines, GFP‐tubulin6 (TUB6)‐, basic proline‐rich protein1 (BPP1)‐GFP‐ and spira1‐like3 (SP1L3)‐GFP‐expressing using a constitutive promoter, and spiral2 (SPR2)‐GFP‐ and GFP‐65 kDa MAP‐1 (MAP65‐1)‐expressing using a native promoter, the length of hypocotyls grown under microgravity conditions in space was longer than that grown at 1 g conditions on the ground. In contrast, the diameter of hypocotyls grown under microgravity conditions was smaller than that of the hypocotyls grown at 1 g. The percentage of cells with transverse microtubules was increased under microgravity conditions, irrespective of the lines. Also, the average angle of the microtubules with respect to the transverse cell axis was decreased in hypocotyls grown under microgravity conditions. When GFP fluorescence was quantified in hypocotyls of GFP‐MAP65‐1 and SPR2‐GFP lines, microgravity increased the levels of MAP65‐1, which appears to be involved in the maintenance of transverse microtubule orientation. However, the levels of SPR2 under microgravity conditions were comparable to those at 1 g. These results suggest that the microgravity‐induced increase in the levels of MAP65‐1 is involved in increase in the transverse microtubules, which may lead to modification of growth anisotropy, thereby developing longer and thinner hypocotyls under microgravity conditions in space.     

On the Origin of Extracellular Glutamate Levels Monitored in the Basal Ganglia of the Rat by In Vivo Microdialysis

Abstract: Several putative neurotransmitters and metabolites were monitored simultaneously in the extracellular space of neostriatum, substantia nigra, and cortex and in subcutaneous tissue of the rat by in vivo microdialysis. Glutamate (Glu) and aspartate (Asp) were at submicromolar and γ-aminobutyric acid (GABA) was at nanomolar concentrations in all brain regions. The highest concentration of dopamine (DA) was in the neostriatum. Dynorphin B (Dyn B) was in the picomolar range in all brain regions. Although no GABA, DA, or Dyn B could be detected in subcutaneous tissue, Glu and Asp levels were ≈5 and ≈0.4 µM, respectively. Lactate and pyruvate concentrations were ≈200 and ≈10 µM in all regions. The following criteria were applied to ascertain the neuronal origin of substances quantified by microdialysis: sensitivity to (a) K⁺ depolarization, (b) Na⁺ channel blockade, (c) removal of extracellular Ca²⁺, and (d) depletion of presynaptic vesicles by local administration of α-latrotoxin. DA, Dyn B, and GABA largely satisfied all these criteria. In contrast, Glu and Asp levels were not greatly affected by K⁺ depolarization and were increased by perfusing with tetrodotoxin or with Ca²⁺-free medium, arguing against a neuronal origin. However, Glu and Asp, as well as DA and GABA, levels were decreased under both basal and K⁺-depolarizing conditions by α-latrotoxin. Because the effect of K⁺ depolarization on Glu and Asp could be masked by reuptake into nerve terminals and glial cells, the reuptake blocker dihydrokainic acid (DHKA) or l -trans-pyrrolidine-2,4-dicarboxylic acid (PDC) was included in the microdialysis perfusion medium. The effect of K⁺ depolarization on Glu and Asp levels was increased by DHKA, but GABA levels were also affected. In contrast, PDC increased only Glu levels. It is concluded that there is a pool of releasable Glu and Asp in the rat brain. However, extracellular levels of amino acids monitored by in vivo microdialysis reflect the balance between neuronal release and reuptake into surrounding nerve terminals and glial elements.     

Increase in synthesis of human monoclonal antibodies by transfected Sp2/0 myeloma mouse cell line under conditions of microgravity

Microgravity can influence cell growth and function. A transfected Sp2/0 myeloma cell line P3A2 producing a human IgG1 anti-TNF monoclonal antibody was cultivated in static culture, spinner flasks and simulated microgravity using a rotating wall vessel bioreactor. Microgravity significantly decreased cell growth (from 1.7×10⁶ to 7.9×10⁵ cells/ml), but facilitated the synthesis of antibodies, (1.8, 1.3 and 0.5 g of anti-TNF hmAb per 10⁶ viable cells for cells cultivated under microgravity, in spinner flasks and static cultures, respectively). The results suggest that microgravity could be applied to improve the specific productivity of cell lines producing potentially important therapeutic proteins.     

Molecular dissection of the Munc18c/syntaxin4 interaction: implications for regulation of membrane trafficking

Sec1p/Munc18 (SM) proteins are believed to play an integral role in vesicle transport through their interaction with SNAREs. Different SM proteins have been shown to interact with SNAREs via different mechanisms, leading to the conclusion that their function has diverged. To further explore this notion, in this study, we have examined the molecular interactions between Munc18c and its cognate SNAREs as these molecules are ubiquitously expressed in mammals and likely regulate a universal plasma membrane trafficking step. Thus, Munc18c binds to monomeric syntaxin4 and the N-terminal 29 amino acids of syntaxin4 are necessary for this interaction. We identified key residues in Munc18c and syntaxin4 that determine the N-terminal interaction and that are consistent with the N-terminal binding mode of yeast proteins Sly1p and Sed5p. In addition, Munc18c binds to the syntaxin4/SNAP23/VAMP2 SNARE complex. Pre-assembly of the syntaxin4/Munc18c dimer accelerates the formation of SNARE complex compared to assembly with syntaxin4 alone. These data suggest that Munc18c interacts with its cognate SNAREs in a manner that resembles the yeast proteins Sly1p and Sed5p rather than the mammalian neuronal proteins Munc18a and syntaxin1a. The Munc18c-SNARE interactions described here imply that Munc18c could play a positive regulatory role in SNARE assembly.     

SNAP-25/syntaxin 1A complex functionally modulates neurotransmitter gamma-aminobutyric acid reuptake

Neurotransmitter gamma-aminobutyric acid (GABA) release to the synaptic clefts is mediated by the formation of a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which includes two target SNAREs syntaxin 1A and SNAP-25 and one vesicle SNARE VAMP-2. The target SNAREs syntaxin 1A and SNAP-25 form a heterodimer, the putative intermediate of the SNARE complex. Neurotransmitter GABA clearance from synaptic clefts is carried out by the reuptake function of its transporters to terminate the postsynaptic signaling. Syntaxin 1A directly binds to the neuronal GABA transporter GAT-1 and inhibits its reuptake function. However, whether other SNARE proteins or SNARE complex regulates GABA reuptake remains unknown. Here we demonstrate that SNAP-25 efficiently inhibits GAT-1 reuptake function in the presence of syntaxin 1A. This inhibition depends on SNAP-25/syntaxin 1A complex formation. The H3 domain of syntaxin 1A is identified as the binding sites for both SNAP-25 and GAT-1. SNAP-25 binding to syntaxin 1A greatly potentiates the physical interaction of syntaxin 1A with GAT-1 and significantly enhances the syntaxin 1A-mediated inhibition of GAT-1 reuptake function. Furthermore, nitric oxide, which promotes SNAP-25 binding to syntaxin 1A to form the SNARE complex, also potentiates the interaction of syntaxin 1A with GAT-1 and suppresses GABA reuptake by GAT-1. Thus our findings delineate a further molecular mechanism for the regulation of GABA reuptake by a target SNARE complex and suggest a direct coordination between GABA release and reuptake.     

Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation

Background

Glutamate and ??-aminobutyric acid (GABA) transporters play important roles in balancing excitatory and inhibitory signals in the brain. Increasing evidence suggest that they may act concertedly to regulate extracellular levels of the neurotransmitters.

Results

Here we present evidence that glutamate uptake-induced release of GABA from astrocytes has a direct impact on the excitability of pyramidal neurons in the hippocampus. We demonstrate that GABA, synthesized from the polyamine putrescine, is released from astrocytes by the reverse action of glial GABA transporter (GAT) subtypes GAT-2 or GAT-3. GABA release can be prevented by blocking glutamate uptake with the non-transportable inhibitor DHK, confirming that it is the glutamate transporter activity that triggers the reversal of GABA transporters, conceivably by elevating the intracellular Na⁺ concentration in astrocytes. The released GABA significantly contributes to the tonic inhibition of neurons in a network activity-dependent manner. Blockade of the Glu/GABA exchange mechanism increases the duration of seizure-like events in the low-[Mg²⁺] in vitro model of epilepsy. Under in vivo conditions the increased GABA release modulates the power of gamma range oscillation in the CA1 region, suggesting that the Glu/GABA exchange mechanism is also functioning in the intact hippocampus under physiological conditions.

Conclusions

The results suggest the existence of a novel molecular mechanism by which astrocytes transform glutamatergic excitation into GABAergic inhibition providing an adjustable, in situ negative feedback on the excitability of neurons.     

Sly1 binds to Golgi and ER syntaxins via a conserved N-terminal peptide motif

Sec1/munc18-like proteins (SM proteins) and SNARE complexes are probably universally required for membrane fusion. However, the molecular mechanism by which they interact has only been defined for synaptic vesicle fusion where munc18 binds to syntaxin in a closed conformation that is incompatible with SNARE complex assembly. We now show that Sly1, an SM protein involved in Golgi and ER fusion, binds to a short, evolutionarily conserved N-terminal peptide of Sed5p and Ufe1p in yeast and of syntaxins 5 and 18 in vertebrates. In these syntaxins, the Sly1 binding peptide is upstream of a separate, autonomously folded N-terminal domain. These data suggest a potentially general mechanism by which SM proteins could interact with peptides in target proteins independent of core complex assembly and suggest that munc18 binding to syntaxin is an exception.