Mitochondrial bioenergetics and cytometric characterization of a synaptosomal preparation from mouse brain cortex.

Mitochondrial function at synapses can be assessed in isolated nerve terminals. Synaptosomes are structures obtained in vitro by detaching the nerve endings from neuronal bodies under controlled homogenization conditions. Several protocols have been described for the preparation of intact synaptosomal fractions. Herein a fast and economical method to obtain synaptosomes with optimal intrasynaptic mitochondria functionality was described. Synaptosomal fractions were obtained from mouse brain cortex by differential centrifugation followed by centrifugation in a Ficoll gradient. The characteristics of the subcellular particles obtained were analyzed by flow cytometry employing specific tools. Integrity and specificity of the obtained organelles were evaluated by calcein and SNAP-25 probes. The proportion of positive events of the synaptosomal preparation was 75 ± 2 % and 48 ± 7% for calcein and Synaptosomal-Associated Protein of 25 kDa (SNAP-25), respectively. Mitochondrial integrity was evaluated by flow cytometric analysis of cardiolipin content, which indicated that 73 ± 1% of the total events were 10 N-nonylacridine orange (NAO)-positive. Oxygen consumption, ATP production and mitochondrial membrane potential determinations showed that mitochondria inside synaptosomes remained functional after the isolation procedure. Mitochondrial and synaptosomal enrichment were determined by measuring synaptosomes/ homogenate ratio of specific markers. Functionality of synaptosomes was verified by nitric oxide detection after glutamate addition. As compared with other methods, the present protocol can be performed briefly, does not imply high economic costs, and provides an useful tool for the isolation of a synaptosomal preparation with high mitochondrial respiratory capacity and an adequate integrity and function of intraterminal mitochondria.

Proteome Profiling of Rat Brain Cortical Changes during Early Postnatal Brain Development.

Label-free quantitation (LFQ) was applied to proteome profiling of rat brain cortical development during the early postnatal period. Male and female rat brain extracts were prepared using a convenient, detergent-free sample preparation technique at postnatal days (PND) 2, 8, 15, and 22. The PND protein ratios were calculated using Proteome Discoverer, and the PND protein change profiles were constructed separately for male and female animals for key presynaptic, postsynaptic, and adhesion brain proteins. The profiles were compared to the analogous profiles assembled from the published mouse and rat cortex proteomic data, including the fractionated-synaptosome data. The PND protein-change trendlines, Pearson correlation coefficient (PCC), and linear regression analysis of the statistically significant PND protein changes were used in the comparative analysis of the datasets. The analysis identified similarities and differences between the datasets. Importantly, there were significant similarities in the comparison of the rat cortex PND (current work) vs mouse (previously published) PND profiles, although in general, a lower abundance of synaptic proteins in mice than in rats was found. The male and female rat cortex PND profiles were expectedly almost identical (98-99% correlation by PCC), which also substantiated this LFQ nanoflow liquid chromatography-high-resolution mass spectrometry approach.

m-Calpain is released from striatal synaptosomes.

Purpose of the study: We aimed to investigate whether m-calpain (a Ca2+-dependent neutral cysteine protease) is released from synaptosomes.Materials and methods: This research was carry on Wistar male rats and isolated nerve endings - synaptosomes. The synaptosomal integrity was checked by the method of measuring LDH activity. Activity of calpains was measured by the casein zymography in gel and in solution. Extracellular calpain was detected by immunoprecipitation and immunoblotting procedures Prediction of secreted proteins peptide on a protein sequence through a local version of the PrediSi tool (http://www.predisi.de). The probability of calpain isoform nonclassical secretion was analyzed by using SecretomeP (http://www.cbs.dtu.dk/services/SecretomeP2.0) software.Results: It has been shown that calcium- and time-dependent m-calpain is released from synaptosomes in an activated form or in a form capable of activation, and this process is not a result of a violation of the integrity of synaptosomes. Analysis of the probability of secretion of the small catalytic subunit of rat m-calpain along a nonclassical pathway showed a high probability of its secretion. Additionally, the release of calpain from synaptosomes revealed by us is suppressed by the addition of glyburide, an ABC transporter inhibitor, to the incubation medium. Among extracellular proteins, potential substrates of calpains are of calpains are found, for example, matrix metalloprotease-2 and -9, alpha-synuclein, etc.Conclusions: Active m-calpain is present in the media generated from striatal synaptosomes. Glyburide prevents m-calpain release from striatal synaptosomes.

HighlightsActive m-calpain is present in the media generated from striatal synaptosomes.Glyburide prevents m-calpain release from striatal synaptosomes.

Isolation of Mitochondria-Associated ER Membranes (MAMs), Synaptic MAMs, and Glycosphingolipid Enriched Microdomains (GEMs) from Brain Tissues and Neuronal Cells.

Subcellular fractionation is a valuable procedure in cell biology to separate and purify various subcellular constituents from one another, i.e., nucleus, cytosol, membranes/organelles, and cytoskeleton. The procedure relies on the use of differential centrifugation of cell and tissue homogenates. Fractionated subcellular organelles may be subjected to additional purification steps that enable the isolation of specific cellular sub-compartments, including interorganellar membrane contact sites. Here we outline a protocol tailored to the isolation of mitochondria, mitochondria-associated ER membranes (MAMs), and glycosphingolipid enriched microdomains (GEMs) from the adult mouse brain, primary neurospheres, and murine embryonic fibroblasts (MEFs). We also provide a detailed protocol for the purification of synaptosomes and their corresponding MAMs .

Synaptophysin controls synaptobrevin-II retrieval via a cryptic C-terminal interaction site.

The accurate retrieval of synaptic vesicle (SV) proteins during endocytosis is essential for the maintenance of neurotransmission. Synaptophysin (Syp) and synaptobrevin-II (SybII) are the most abundant proteins on SVs. Neurons lacking Syp display defects in the activity-dependent retrieval of SybII and a general slowing of SV endocytosis. To determine the role of the cytoplasmic C terminus of Syp in the control of these two events, we performed molecular replacement studies in primary cultures of Syp knockout neurons using genetically encoded reporters of SV cargo trafficking at physiological temperatures. Under these conditions, we discovered, 1) no slowing in SV endocytosis in Syp knockout neurons, and 2) a continued defect in SybII retrieval in knockout neurons expressing a form of Syp lacking its C terminus. Sequential truncations of the Syp C-terminus revealed a cryptic interaction site for the SNARE motif of SybII that was concealed in the full-length form. This suggests that a conformational change within the Syp C terminus is key to permitting SybII binding and thus its accurate retrieval. Furthermore, this study reveals that the sole presynaptic role of Syp is the control of SybII retrieval, since no defect in SV endocytosis kinetics was observed at physiological temperatures.

Quantitative proteomics of synaptosome S-nitrosylation in Alzheimer's disease.

Increasing evidence suggests that both synaptic loss and neuroinflammation constitute early pathologic hallmarks of Alzheimer's disease. A downstream event during inflammatory activation of microglia and astrocytes is the induction of nitric oxide synthase type 2, resulting in an increased release of nitric oxide and the post-translational S-nitrosylation of protein cysteine residues. Both early events, inflammation and synaptic dysfunction, could be connected if this excess nitrosylation occurs on synaptic proteins. In the long term, such changes could provide new insight into patho-mechanisms as well as biomarker candidates from the early stages of disease progression. This study investigated S-nitrosylation in synaptosomal proteins isolated from APP/PS1 model mice in comparison to wild type and NOS2-/- mice, as well as human control, mild cognitive impairment and Alzheimer's disease brain tissues. Proteomics data were obtained using an established protocol utilizing an isobaric mass tag method, followed by nanocapillary high performance liquid chromatography tandem mass spectrometry. Statistical analysis identified the S-nitrosylation sites most likely derived from an increase in nitric oxide (NO) in dependence of presence of AD pathology, age and the key enzyme NOS2. The resulting list of candidate proteins is discussed considering function, previous findings in the context of neurodegeneration, and the potential for further validation studies.

Critical Analysis of Particle Detection Artifacts in Synaptosome Flow Cytometry.

Flow cytometry and fluorescence-activated sorting are powerful techniques that hold great promise for studying heterogeneous populations of submicron particles such as synaptosomes, but many technical challenges arise in these experiments. To date, most flow cytometry studies of synaptosomes have relied on particle detection using forward scatter (FSC) measurements and size estimation with polystyrene (PS) bead standards. However, these practices have serious limitations, and special care must be taken to overcome the poor sensitivity of conventional flow cytometers in the analysis of submicron particles. Technical artifacts can confound these experiments, especially the detection of multiple particles as a single event. Here, we compared analysis of P2 crude synaptosomal preparations from murine forebrain on multiple flow cytometers using both FSC-triggered and fluorescence-triggered detection. We implemented multicolor fluorescent dye-based assays to quantify coincident particle detection and aggregation, and we assessed the false colocalization of antigens in immunostaining analyses. Our results demonstrate that fluorescence triggering and proper dilution can control for coincident particle detection, but not particle aggregation. We confirmed previous studies showing that FSC-based size estimation with PS beads underestimates biological particle size, and we identified pervasive aggregation in the FSC range analyzed in most synaptosome flow cytometry studies. We found that analyzing P2 samples in sucrose/EDTA/tris (SET) buffer reduces aggregation compared to PBS, but does not completely eliminate the presence of aggregates, especially in immunostaining experiments. Our study highlights challenges and pitfalls in synaptosome flow cytometry and provides a methodological framework for future studies.

Branched Sialylated N-glycans Are Accumulated in Brain Synaptosomes and Interact with Siglec-H.

Proper N-glycosylation of proteins is important for normal brain development and nervous system function. Identification of the localization, carrier proteins and interacting partners of N-glycans is essential for understanding the roles of glycoproteins. The present study examined the N-glycan A2G'2F (Galß1-3GlcNAcß1-2Manα1-6[Galß1-3GlcNAcß1-2Manα1-3]Manß1-4GlcNAcß1-4[Fucα1-6]GlcNAc-). A2G'2F has a branched sialic acid structural feature, and branched sialylated A2G'2F is a major N-glycan in the mouse brain. Its expression in the mouse brain increases during development, suggesting that branched sialylated N-glycans play essential roles during brain development. However, the carrier proteins, interacting partners and localization of branched sialylated N-glycans remain unknown. We previously improved our method for analyzing N-glycans from trace samples, and here we succeeded in detecting A2G'2F in small fragments excised from the two-dimensional electrophoresis gels of subcellular fractionated mouse brain proteins. A2G'2F was accumulated in mouse brain synaptosomes. We identified calreticulin as one of the candidate A2G'2F carriers and found calreticulin expression in both the endoplasmic reticulum and synaptosomal fractions. Calreticulin was observed in dendritic spines of cultured cortical neurons. Synthesized branched sialylated glycan clusters interacted with sialic acid-binding immunoglobulin-like lectin H (Siglec-H), which is known to be a microglia-specific molecule. Taken together, these results suggest that branched sialylated A2G'2F in synaptosomes plays a role in the interaction of dendritic spines with microglia.Key words: N-glycan, subcellular fractionation, calreticulin, dendritic spine, Siglec-H.

Preparation of polysomal fractions from mouse brain synaptoneurosomes and analysis of polysomal-bound mRNAs.

BACKGROUND: Here we describe a detailed, reliable protocol for isolation of polysomal fractions from mouse brain synaptoneurosomes. This method is an important tool to study local protein synthesis in neurons. NEW METHOD: We combined rapid preparation of synaptoneurosomes by filtration with polysome profiling. We provide a detailed protocol highlighting difficulties and critical steps of: i) preparation of synaptoneurosomes; ii) polyribosome fractionation from synaptoneurosomes; iii) extraction of proteins and RNA from sucrose gradient fractions. RESULTS: and Comparison with Existing Methods We fractionated polyribosomes from synaptoneurosomes and detected the association of Mmp9, Camk2a and Stx1B mRNA with polysomes in the unstimulated conditions. Synaptic stimulation led to increased levels of Mmp9 and Camk2a mRNA in the heavy polysomal fractions. We compared our protocol with existing methods CONCLUSIONS: We have developed a reliable, effective method to prepare polyribosomal fractions from synaptoneurosomes to study polyribosomal binding of mRNAs as an aspect of synaptic translation in vitro.

Synaptosomal Proteome of the Orbitofrontal Cortex from Schizophrenia Patients Using Quantitative Label-Free and iTRAQ-Based Shotgun Proteomics.

Schizophrenia is a chronic and incurable neuropsychiatric disorder that affects about one percent of the world population. The proteomic characterization of the synaptosome fraction of the orbitofrontal cortex is useful for providing valuable information about the molecular mechanisms of synaptic functions in these patients. Quantitative analyses of synaptic proteins were made with eight paranoid schizophrenia patients and a pool of eight healthy controls free of mental diseases. Label-free and iTRAQ labeling identified a total of 2018 protein groups. Statistical analyses revealed 12 and 55 significantly dysregulated proteins by iTRAQ and label-free, respectively. Quantitative proteome analyses showed an imbalance in the calcium signaling pathway and proteins such as reticulon-1 and cytochrome c, related to endoplasmic reticulum stress and programmed cell death. Also, it was found that there is a significant increase in limbic-system-associated membrane protein and α-calcium/calmodulin-dependent protein kinase II, associated with the regulation of human behavior. Our data contribute to a better understanding about apoptosis as a possible pathophysiological mechanism of this disease as well as neural systems supporting social behavior in schizophrenia. This study also is a joint effort of the Chr 15 C-HPP team and the Human Brain Proteome Project of B/D-HPP. All MS proteomics data are deposited in the ProteomeXchange Repository under PXD006798.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization.

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

The sigma-1 receptor modulates dopamine transporter conformation and cocaine binding and may thereby potentiate cocaine self-administration in rats.

The dopamine transporter (DAT) regulates dopamine (DA) neurotransmission by recapturing DA into the presynaptic terminals and is a principal target of the psychostimulant cocaine. The sigma-1 receptor (σ1R) is a molecular chaperone, and its ligands have been shown to modulate DA neuronal signaling, although their effects on DAT activity are unclear. Here, we report that the prototypical σ1R agonist (+)-pentazocine potentiated the dose response of cocaine self-administration in rats, consistent with the effects of the σR agonists PRE-084 and DTG (1,3-di-o-tolylguanidine) reported previously. These behavioral effects appeared to be correlated with functional changes of DAT. Preincubation with (+)-pentazocine or PRE-084 increased the Bmax values of [3H]WIN35428 binding to DAT in rat striatal synaptosomes and transfected cells. A specific interaction between σ1R and DAT was detected by co-immunoprecipitation and bioluminescence resonance energy transfer assays. Mutational analyses indicated that the transmembrane domain of σ1R likely mediated this interaction. Furthermore, cysteine accessibility assays showed that σ1R agonist preincubation potentiated cocaine-induced changes in DAT conformation, which were blocked by the specific σ1R antagonist CM304. Moreover, σ1R ligands had distinct effects on σ1R multimerization. CM304 increased the proportion of multimeric σ1Rs, whereas (+)-pentazocine increased monomeric σ1Rs. Together these results support the hypothesis that σ1R agonists promote dissociation of σ1R multimers into monomers, which then interact with DAT to stabilize an outward-facing DAT conformation and enhance cocaine binding. We propose that this novel molecular mechanism underlies the behavioral potentiation of cocaine self-administration by σ1R agonists in animal models.

[Effect of baicalin on ATPase and LDH and its regulatory effect on the AC/cAMP/PKA signaling pathway in rats with attention deficit hyperactivity disorder].

OBJECTIVE: To study the effect of baicalin on synaptosomal adenosine triphosphatase (ATPase) and lactate dehydrogenase (LDH) and its regulatory effect on the adenylate cyclase (AC)/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway in rats with attention deficit hyperactivity disorder (ADHD). METHODS: A total of 40 SHR rats were randomly divided into five groups: ADHD model, methylphenidate hydrochloride treatment (0.07 mg/mL), and low-dose (3.33 mg/mL), medium-dose (6.67 mg/mL), and high-dose (10 mg/mL) baicalin treatment (n=8 each). Eight WKY rats were selected as normal control group. Percoll density gradient centrifugation was used to prepare brain synaptosomes and an electron microscope was used to observe their structure. Colorimetry was used to measure the activities of ATPase and LDH in synaptosomes. ELISA was used to measure the content of AC, cAMP, and PKA. RESULTS: Compared with the normal control group, the ADHD model group had a significant reduction in the ATPase activity, a significant increase in the LDH activity, and significant reductions in the content of AC, cAMP, and PKA (P<0.05). Compared with the ADHD model group, the methylphenidate hydrochloride group and the medium- and high-dose baicalin groups had a significant increase in the ATPase activity (P<0.05), a significant reduction in the LDH activity (P<0.05), and significant increases in the content of AC, cAMP, and PKA (P<0.05). Compared with the methylphenidate hydrochloride group, the high-dose baicalin group had significantly greater changes in these indices (P<0.05). Compared with the low-dose baicalin group, the high-dose baicalin group had a significant increase in the ATPase activity (P<0.05); the medium- and high-dose baicalin groups had a significant reduction in the LDH activity (P<0.05) and significant increases in the content of AC, cAMP, and PKA (P<0.05). Compared with the medium-dose baicalin group, the high-dose baicalin group had a significant increase in the ATPase activity (P<0.05). CONCLUSIONS: Both methylphenidate hydrochloride and baicalin can improve synaptosomal ATPase and LDH activities in rats with ADHD. The effect of baicalin is dose-dependent, and high-dose baicalin has a significantly greater effect than methylphenidate hydrochloride. Baicalin exerts its therapeutic effect possibly by upregulating the AC/cAMP/PKA signaling pathway.

Unique Lipid Chemistry of Synaptic Vesicle and Synaptosome Membrane Revealed Using Mass Spectrometry.

Synaptic vesicles measuring 30-50 nm in diameter containing neurotransmitters either completely collapse at the presynaptic membrane or dock and transiently fuse at the base of specialized 15 nm cup-shaped lipoprotein structures called porosomes at the presynaptic membrane of synaptosomes to release neurotransmitters. Recent study reports the unique composition of major lipids associated with neuronal porosomes. Given that lipids greatly influence the association and functions of membrane proteins, differences in lipid composition of synaptic vesicle and the synaptosome membrane was hypothesized. To test this hypothesis, the lipidome of isolated synaptosome, synaptosome membrane, and synaptic vesicle preparation were determined by using mass spectrometry in the current study. Results from the study demonstrate the enriched presence of triacyl glycerols and sphingomyelins in synaptic vesicles, as opposed to the enriched presence of phospholipids in the synaptosome membrane fraction, reflecting on the tight regulation of nerve cells in compartmentalization of membrane lipids at the nerve terminal.

Comparison of simple sucrose and percoll based methodologies for synaptosome enrichment.

Synaptosomes are isolated nerve terminals. They represent an extremely attractive in vitro model system to study synaptic physiology since they preserve morphological and functional characteristics of the synapse. As such they have been used to investigate synaptic dysfunctions associated with neuropathologies like Alzheimer's disease. In the present work two simple methodologies for isolating synaptosomal-enriched fractions were compared for the first time. The starting points of both protocols were rat cortical or hippocampal homogenized tissues that underwent several differential centrifugation steps followed by a final purification of synaptosomal-enriched fractions using either a Percoll gradient or a Sucrose gradient. Comparison of the fractions obtained was carried out, using both biochemical and electron microscopy approaches. In the biochemical analysis the protein levels of pre-synaptic, post-synaptic, nuclear and mitochondrial markers were evaluated. Additional characterization of the synaptosomal-enriched fractions was performed using transmission electron microscopy. In summary, the results indicate that under the conditions tested the Sucrose based protocol is more efficient for the isolation of synaptosomal-enriched fractions from both neuronal tissues, being particularly efficient for hippocampus that is a less abundant brain tissue. Further, the sucrose protocol apparently results in a higher yield of viable synaptosomes suitable for further assays, including structural and functional studies of synapses; making this an attractive procedure to study processes associated with neuropathologies.

Mass spectrometric analysis of synaptosomal membrane preparations for the determination of brain receptors, transporters and channels.

The molecular composition of synaptic signal transduction machineries shapes synaptic neurotransmission. The repertoire of receptors, transporters and channels (RTCs) comprises major signaling events in the brain. RTCs are conventionally studied by candidate immunohistochemistry and biochemistry, which are low throughput with resolution greatly affected by available immunoreagents and membrane interference. Therefore, a comprehensive resource of synaptic brain RTCs is still lacking. In particular, studies on the detergent-soluble synaptosomal fraction, known to contain transporters and channels, are limited. We, therefore, performed sub-synaptosomal fractionation of rat cerebral cortex, followed by trypsin/chymotrypsin sequential digestion of a detergent-soluble synaptosomal fraction and a postsynaptic density preparation, stable-isotope tryptic peptide labeling and liquid chromatography mass spectrometry. Based on the current study, a total of 4784 synaptic proteins were submitted to the ProteomExchange database (PXD001948), including 274 receptors, 394 transporters/channels and 1377 transmembrane proteins. Function-based classification assigned 1781 proteins as probable drug targets with 834 directly linked to brain disorders. The analytical approach identified 499 RTCs that are not listed in the largest, curated database for synaptosomal proteins (SynProt). This is a threefold RTC increase over all other data collected to date. Taken together, we present a protein discovery resource that can serve as a benchmark for future molecular interrogation of synaptic connectivity.

A Novel Bifunctional Alkylphenol Anesthetic Allows Characterization of γ-Aminobutyric Acid, Type A (GABAA), Receptor Subunit Binding Selectivity in Synaptosomes.

Propofol, an intravenous anesthetic, is a positive modulator of the GABAA receptor, but the mechanistic details, including the relevant binding sites and alternative targets, remain disputed. Here we undertook an in-depth study of alkylphenol-based anesthetic binding to synaptic membranes. We designed, synthesized, and characterized a chemically active alkylphenol anesthetic (2-((prop-2-yn-1-yloxy)methyl)-5-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenol, AziPm-click (1)), for affinity-based protein profiling (ABPP) of propofol-binding proteins in their native state within mouse synaptosomes. The ABPP strategy captured ∼4% of the synaptosomal proteome, including the unbiased capture of five α or ß GABAA receptor subunits. Lack of γ2 subunit capture was not due to low abundance. Consistent with this, independent molecular dynamics simulations with alchemical free energy perturbation calculations predicted selective propofol binding to interfacial sites, with higher affinities for α/ß than γ-containing interfaces. The simulations indicated hydrogen bonding is a key component leading to propofol-selective binding within GABAA receptor subunit interfaces, with stable hydrogen bonds observed between propofol and α/ß cavity residues but not γ cavity residues. We confirmed this by introducing a hydrogen bond-null propofol analogue as a protecting ligand for targeted-ABPP and observed a lack of GABAA receptor subunit protection. This investigation demonstrates striking interfacial GABAA receptor subunit selectivity in the native milieu, suggesting that asymmetric occupancy of heteropentameric ion channels by alkylphenol-based anesthetics is sufficient to induce modulation of activity.

Pentobarbital modifies the lipid raft-protein interaction: A first clue about the anesthesia mechanism on NMDA and GABAA receptors.

Recent studies have shown that anesthetic agents alter the physical properties of lipid rafts on model membranes. However, if this destabilization occurs in brain membranes, altering the lipid raft-protein interaction, remains unknown. We analyzed the effects produced by pentobarbital (PB) on brain plasma membranes and lipid rafts in vivo. We characterized for the first time the thermotropic behavior of plasma membranes, synaptosomes, and lipid rafts from rat brain. We found that the transition temperature from the ordered gel to disordered liquid phase of lipids is close to physiological temperature. We then studied the effect of PB on protein composition of lipid rafts. Our results show a reduction of the total protein associated to rafts, with a higher reduction of the NMDAR compared to the GABAA receptor. Both receptors are considered the main targets of PB. In general, our results suggest that lipid rafts could be plausible mediators in anesthetic action.

Quantitative Proteome Profiling of Street Rabies Virus-Infected Mouse Hippocampal Synaptosomes.

It is well established now that neuronal dysfunction rather than structural damage may be responsible for the development of rabies. In order to explore the underlying mechanisms in rabies virus (RABV) and synaptic dysfunctions, a quantitative proteome profiling was carried out on synaptosome samples from mice hippocampus. Synaptosome samples from mice hippocampus were isolated and confirmed by Western blot and transmission electron microscopy. Synaptosome protein content changes were quantitatively detected by Nano-LC-MS/MS. Protein functions were classified by the Gene Ontology (GO) and KEGG pathway. PSICQUIC was used to create a network. MCODE algorithm was applied to obtain subnetworks. Of these protein changes, 45 were upregulated and 14 were downregulated following RABV infection relative to non-infected (mock) synaptosomes. 28 proteins were unique to mock treatment and 12 were unique to RABV treatment. Proteins related to metabolism and synaptic vesicle showed the most changes in expression levels. Furthermore, protein-protein interaction (PPI) networks revealed that several key biological processes related to synaptic functions potentially were modulated by RABV, including energy metabolism, cytoskeleton organization, and synaptic transmission. These data will be useful for better understanding of neuronal dysfunction of rabies and provide the foundation for future research.

Xanthohumol-induced presynaptic reduction of glutamate release in the rat hippocampus.

This study examined whether xanthohumol, a hop-derived prenylated flavonoid present in beer, affects glutamate release in the rat hippocampus. In the rat hippocampal nerve terminals (synaptosomes), xanthohumol inhibited the release of 4-aminopyridine (4-AP)-evoked glutamate and the elevation of cytosolic Ca(2+) concentration, whereas it had no effect on 4-AP-mediated depolarization. The inhibitory effect of xanthohumol on the evoked glutamate release was prevented by removing extracellular Ca(2+), using the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channel blocker ω-CgTX MVIIC, the calmodulin antagonists W7 and calmidazolium, and the protein kinase A inhibitor H89; however, no such effect was observed when the G-protein inhibitor N-ethylmaleimide was used. In addition, immunocytochemical data demonstrated that GABAA receptors are present in the hippocampal synaptosomes and that the xanthohumol effect on evoked glutamate release was antagonized by the GABAA receptor antagonist SR95531. Furthermore, in slice preparations, xanthohumol reduced the frequency of miniature excitatory postsynaptic currents without affecting their amplitude. We conclude that xanthohumol acts at GABAA receptors present in the hippocampal nerve terminals to decrease the Ca(2+) influx through N- and P/Q-type Ca(2+) channels, which subsequently suppresses the Ca(2+)-calmodulin/PKA cascade to decrease the evoked glutamate release.