Super-Resolution Imaging of Tau Proteins in Isolated and Immobilized Brain Synaptosomes.

Tau protein has important physiological functions at both presynaptic and postsynaptic terminals. Pathological tau species are also associated with synaptic dysfunctions in several neurodegenerative disorders, especially Alzheimer's disease. To understand tau distribution inside synaptic compartments, super-resolution imaging is required. Here, we describe a facile protocol to immobilize and image brain synaptosomes without aggregation artefacts, by substituting the standard fixative paraformaldehyde with ethylene glycol bis(succinimidyl succinate) (EGS). Super-resolution imaging of tau proteins is achieved through three-color direct stochastic optical reconstruction microscopy (dSTORM). Tau protein is found to colocalize with synaptic vesicles as well as postsynaptic densities.

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.

The proteomic landscape of synaptic diversity across brain regions and cell types.

Neurons build synaptic contacts using different protein combinations that define the specificity, function, and plasticity potential of synapses; however, the diversity of synaptic proteomes remains largely unexplored. We prepared synaptosomes from 7 different transgenic mouse lines with fluorescently labeled presynaptic terminals. Combining microdissection of 5 different brain regions with fluorescent-activated synaptosome sorting (FASS), we isolated and analyzed the proteomes of 18 different synapse types. We discovered ∼1,800 unique synapse-type-enriched proteins and allocated thousands of proteins to different types of synapses (https://syndive.org/). We identify shared synaptic protein modules and highlight the proteomic hotspots for synapse specialization. We reveal unique and common features of the striatal dopaminergic proteome and discover the proteome signatures that relate to the functional properties of different interneuron classes. This study provides a molecular systems-biology analysis of synapses and a framework to integrate proteomic information for synapse subtypes of interest with cellular or circuit-level experiments.

A method to study human synaptic protein-protein interactions by using flow cytometry coupled to proximity ligation assay (Syn-FlowPLA).

BACKGROUND: Synapses are highly specialized sites characterized by intricate networks of protein-protein interactions (PPIs) important to maintain healthy synapses. Therefore, mapping these networks could address unsolved questions about human cognition, synaptic plasticity, learning, and memory in physiological and pathological conditions. The limitation of analyzing synaptic interactions in living humans has led to the development of methods to isolate synaptic terminals (synaptosomes) from cryopreserved human brains. NEW METHOD: Here, we established a method to detect synaptic PPIs by applying flow cytometric proximity ligation assay (FlowPLA) to synaptosomes isolated from frozen human frontal cortex (FC) and hippocampus (HP) (Syn-FlowPLA). RESULTS: Applying this method in synaptosomes, we were able to detect the known post-synaptic interactions between distinct subtypes of N-methyl-D-aspartate glutamate receptors (NMDARs) and their anchoring postsynaptic density 95 protein (PSD95). Moreover, we detected the known pre-synaptic interactions between the SNARE complex proteins synaptosomal-associated protein of 25 kDa (SNAP25), synaptobrevin (VAMP2), and syntaxin 1a (STX1A). As a negative control, we analyzed the interaction between mitochondrial superoxide dismutase 2 (SOD2) and PSD95, which are not expected to be physically associated. COMPARISON WITH EXISTING METHODS: PPIs have been studied in vitro primarily by co-immunoprecipitation, affinity chromatography, protein-fragment complementation assays (PCAs), and flow cytometry. All these are valid approaches; however, they require more steps or combination with other techniques. PLA technology identifies PPIs with high specificity and sensitivity. CONCLUSIONS: The Syn-FlowPLA described here allows rapid analyses of PPIs, specifically within the synaptic compartment isolated from frozen autopsy specimens, achieving greater target sensitivity. Syn-FlowPLA, as presented here, is therefore a useful method to study human synaptic PPI in physiological and pathological conditions.

Monoacylglycerol Lipase Inhibition Prevents Short-Term Mitochondrial Dysfunction and Oxidative Damage in Rat Brain Synaptosomal/Mitochondrial Fractions and Cortical Slices: Role of Cannabinoid Receptors.

Inhibition of enzymes responsible for endocannabinoid hydrolysis represents an invaluable emerging tool for the potential treatment of neurodegenerative disorders. Monoacylglycerol lipase (MAGL) is the enzyme responsible for degrading 2-arachydonoylglycerol (2-AG), the most abundant endocannabinoid in the central nervous system (CNS). Here, we tested the effects of the selective MAGL inhibitor JZL184 on the 3-nitropropinic acid (3-NP)-induced short-term loss of mitochondrial reductive capacity/viability and oxidative damage in rat brain synaptosomal/mitochondrial fractions and cortical slices. In synaptosomes, while 3-NP decreased mitochondrial function and increased lipid peroxidation, JZL184 attenuated both markers. The protective effects evoked by JZL184 on the 3-NP-induced mitochondrial dysfunction were primarily mediated by activation of cannabinoid receptor 2 (CB2R), as evidenced by their inhibition by the selective CB2R inverse agonist JTE907. The cannabinoid receptor 1 (CB1R) also participated in this effect in a lesser extent, as evidenced by the CB1R antagonist/inverse agonist AM281. In contrast, activation of CB1R, but not CB2R, was responsible for the protective effects of JZL184 on the 3-NP-iduced lipid peroxidation. Protective effects of JZL184 were confirmed in other toxic models involving excitotoxicity and oxidative damage as internal controls. In cortical slices, JZL184 ameliorated the 3-NP-induced loss of mitochondrial function, the increase in lipid peroxidation, and the inhibition of succinate dehydrogenase (mitochondrial complex II) activity, and these effects were independent on CB1R and CB2R, as evidenced by the lack of effects of AM281 and JTE907, respectively. Our novel results provide experimental evidence that the differential protective effects exerted by JZL184 on the early toxic effects induced by 3-NP in brain synaptosomes and cortical slices involve MAGL inhibition, and possibly the subsequent accumulation of 2-AG. These effects involve pro-energetic and redox modulatory mechanisms that may be either dependent or independent of cannabinoid receptors' activation.

α-Synuclein colocalizes with AP180 and affects the size of clathrin lattices.

α-Synuclein and family members ß- and γ-synuclein are presynaptic proteins that sense and generate membrane curvature, properties important for synaptic vesicle (SV) cycling. αßγ-synuclein triple knockout neurons exhibit SV endocytosis deficits. Here, we investigated if α-synuclein affects clathrin assembly in vitro. Visualizing clathrin assembly on membranes using a lipid monolayer system revealed that α-synuclein increases clathrin lattices size and curvature. On cell membranes, we observe that α-synuclein is colocalized with clathrin and its adapter AP180 in a concentric ring pattern. Clathrin puncta that contain both α-synuclein and AP180 were significantly larger than clathrin puncta containing either protein alone. We determined that this effect occurs in part through colocalization of α-synuclein with the phospholipid PI(4,5)P2 in the membrane. Immuno-electron microscopy (EM) of synaptosomes uncovered that α-synuclein relocalizes from SVs to the presynaptic membrane upon stimulation, positioning α-synuclein to function on presynaptic membranes during or after stimulation. Additionally, we show that deletion of synucleins impacts brain-derived clathrin-coated vesicle size. Thus, α-synuclein affects the size and curvature of clathrin structures on membranes and functions as an endocytic accessory protein.

Mangiferin depresses vesicular glutamate release in synaptosomes from the rat cerebral cortex by decreasing synapsin I phosphorylation.

Mangiferin is a glucosyl xanthone that has been shown to be a neuroprotective agent against brain disorders involving excess glutamate. However, the effect of mangiferin on the function of the glutamatergic system has not been investigated. In this study, we used synaptosomes from the rat cerebral cortex to investigate the effect of mangiferin on glutamate release and identify the possible underlying mechanism. We observed that mangiferin produced a concentration-dependent reduction in the release of glutamate elicited by 4-aminopyridine with an IC50 value of 25 µM. Inhibition of glutamate release was blocked by removing extracellular calcium and by treatment with the vacuolar-type H+-ATPase inhibitor bafilomycin A1, which prevents the uptake and storage of glutamate in vesicles. Moreover, we showed that mangiferin decreased the 4-aminopyridine-elicited FM1-43 release and synaptotagmin 1 luminal domain antibody (syt1-L ab) uptake from synaptosomes, which correlated with decreased synaptic vesicle exocytosis. Transmission electron microscopy in synaptosomes also showed that mangiferin attenuated the 4-aminopyridine-elicited decrease in the number of synaptic vesicles. In addition, antagonism of Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase A (PKA) counteracted mangiferin's effect on glutamate release. Mangiferin also decreased the phosphorylation of CaMKII, PKA, and synapsin I elicited by 4-aminopyridine treatment. Our data suggest that mangiferin reduces PKA and CaMKII activation and synapsin I phosphorylation, which could decrease synaptic vesicle availability and lead to a subsequent reduction in vesicular glutamate release from synaptosomes.

Cynarin, a caffeoylquinic acid derivative in artichoke, inhibits exocytotic glutamate release from rat cortical nerve terminals (synaptosomes).

The purpose of this study was to evaluate the effect of cynarin, a caffeoylquinic acid derivative in artichoke, on glutamate release elicited by 4-aminopyridine (4-AP) in rat cortical nerve terminals (synaptosomes). We observed that cynarin decreased 4-aminopyridine-elicited glutamate release, which was prevented by the removal of external free Ca2+ with ethylene glycol bis (ß-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) or the blockade of P/Q-type calcium channels with ω-agatoxin IVA. Molecular docking also revealed that cynarin formed a hydrogen bond with the P/Q-type Ca2+ channel, indicating a mechanism of action involving Ca2+ influx inhibition. Additionally, the inhibitory effect of cynarin on glutamate release is associated with a change in the available synaptic vesicles, as cynarin decreased 4-AP-elicited FM1-43 release or hypertonic sucrose-evoked glutamate release from synaptosomes. Furthermore, the suppression of protein kinase A (PKA) prevented the effect of cynarin on 4-AP-elicited glutamate release. 4-AP-elicited PKA and synapsin I or synaptosomal-associated protein of 25 kDa (SNAP-25) phosphorylation at PKA-specific residues were also attenuated by cynarin. Our data indicate that cynarin, through the suppression of P/Q-type Ca2+ channels, inhibits PKA activation and attenuates synapsin I and SNAP-25 phosphorylation at PKA-specific residues, thus decreasing synaptic vesicle availability and contributing to glutamate release inhibition in cerebral cortex terminals.

T Cell Immunological Synaptosomes: Definition and Isolation.

In addition to microvilli's role as structural scaffold for TCR clustering, we recently discovered a novel function as message senders. We found that microvilli are separated from the T cell body shortly upon TCR stimulation and vesiculated to form T cell microvilli particles (TMPs), a new type of membrane vesicles. TMPs and synaptic ectosomes, which bud from the synaptic cleft, constitute "T cell immunological synaptosomes (TISs)" and act as conveyors of T cell messages or traits to cognate antigen-presenting cells. In practice, it is almost impossible to distinguish between TMPs and synaptic ectosomes. Here, we describe a newly developed protocol to isolate TISs from activated T cells using antibody-immobilized agarose beads and density gradient ultracentrifugation. We further describe the methods for TIS quantification with flow cytometry and to evaluate TIS efficacy on dendritic cells.

Increased Synaptic ATP Release and CD73-Mediated Formation of Extracellular Adenosine in the Control of Behavioral and Electrophysiological Modifications Caused by Chronic Stress.

Increased ATP release and its extracellular catabolism through CD73 (ecto-5'-nucleotidase) lead to the overactivation of adenosine A2A receptors (A2AR), which occurs in different brain disorders. A2AR blockade blunts mood and memory dysfunction caused by repeated stress, but it is unknown if increased ATP release coupled to CD73-mediated formation of extracellular adenosine is responsible for A2AR overactivation upon repeated stress. This was now investigated in adult rats subject to repeated stress for 14 consecutive days. Frontocortical and hippocampal synaptosomes from stressed rats displayed an increased release of ATP upon depolarization, coupled to an increased density of vesicular nucleotide transporters and of CD73. The continuous intracerebroventricular delivery of the CD73 inhibitor α,ß-methylene ADP (AOPCP, 100 µM) during restraint stress attenuated mood and memory dysfunction. Slice electrophysiological recordings showed that restraint stress decreased long-term potentiation both in prefrontocortical layer II/III-layer V synapses and in hippocampal Schaffer fibers-CA1 pyramid synapses, which was prevented by AOPCP, an effect occluded by adenosine deaminase and by the A2AR antagonist SCH58261. These results indicate that increased synaptic ATP release coupled to CD73-mediated formation of extracellular adenosine contributes to mood and memory dysfunction triggered by repeated restraint stress. This prompts considering interventions decreasing ATP release and CD73 activity as novel strategies to mitigate the burden of repeated stress.

Alterations in the Proteome of Developing Neocortical Synaptosomes in the Absence of MET Signaling Revealed by Comparative Proteomics.

Alterations in the expression of genes encoding proteins involved in synapse formation, maturation, and function are a hallmark of many neurodevelopmental and psychiatric disorders. For example, there is reduced neocortical expression of the MET receptor tyrosine kinase (MET) transcript and protein in Autism Spectrum Disorder (ASD) and Rett syndrome. Preclinical in vivo and in vitro models manipulating MET signaling reveal that the receptor modulates excitatory synapse development and maturation in select forebrain circuits. The molecular adaptations underlying the altered synaptic development remain unknown. We performed a comparative mass spectrometry analysis of synaptosomes generated from the neocortex of wild type and Met null mice during the peak of synaptogenesis (postnatal day 14; data are available from ProteomeXchange with identifier PXD033204). The analyses revealed broad disruption of the developing synaptic proteome in the absence of MET, consistent with the localization of MET protein in pre- and postsynaptic compartments, including proteins associated with the neocortical synaptic MET interactome and those encoded by syndromic and ASD risk genes. In addition to an overrepresentation of altered proteins associated with the SNARE complex, multiple proteins in the ubiquitin-proteasome system and associated with the synaptic vesicle, as well as proteins that regulate actin filament organization and synaptic vesicle exocytosis/endocytosis, were disrupted. Taken together, the proteomic changes are consistent with structural and functional changes observed following alterations in MET signaling. We hypothesize that the molecular adaptations following Met deletion may reflect a general mechanism that produces circuit-specific molecular changes due to loss or reduction of synaptic signaling proteins.

Plantainoside D Reduces Depolarization-Evoked Glutamate Release from Rat Cerebral Cortical Synaptosomes.

Inhibiting the excessive release of glutamate in the brain is emerging as a promising therapeutic option and is efficient for treating neurodegenerative disorders. The aim of this study is to investigate the effect and mechanism of plantainoside D (PD), a phenylenthanoid glycoside isolated from Plantago asiatica L., on glutamate release in rat cerebral cortical nerve terminals (synaptosomes). We observed that PD inhibited the potassium channel blocker 4-aminopyridine (4-AP)-evoked release of glutamate and elevated concentration of cytosolic Ca2+. Using bafilomycin A1 to block glutamate uptake into synaptic vesicles and EDTA to chelate extracellular Ca2+, the inhibitory effect of PD on 4-AP-evoked glutamate release was prevented. In contrast, the action of PD on the 4-AP-evoked release of glutamate in the presence of dl-TBOA, a potent nontransportable inhibitor of glutamate transporters, was unaffected. PD does not alter the 4-AP-mediated depolarization of the synaptosomal membrane potential, suggesting that the inhibitory effect of PD on glutamate release is associated with voltage-dependent Ca2+ channels (VDCCs) but not the modulation of plasma membrane potential. Pretreatment with the Ca2+ channel blocker (N-type) ω-conotoxin GVIA abolished the inhibitory effect of PD on the evoked glutamate release, as did pretreatment with the protein kinase C inhibitor GF109203x. However, the PD-mediated inhibition of glutamate release was eliminated by applying the mitochondrial Na+/Ca2+ exchanger inhibitor CGP37157 or dantrolene, which inhibits Ca2+ release through ryanodine receptor channels. These data suggest that PD mediates the inhibition of evoked glutamate release from synaptosomes primarily by reducing the influx of Ca2+ through N-type Ca2+ channels, subsequently reducing the protein kinase C cascade.

Fast and efficient synaptosome isolation and post-synaptic density enrichment from hiPSC-motor neurons by biochemical sub-cellular fractionation.

We describe here a time-efficient, in-house protocol for synaptosome isolation and enrichment of the post-synaptic density (PSD) from hiPSC-derived motor neurons. By using biochemical sub-cellular fractionation, the crude synaptosome is first isolated from the cytosol and is then further separated into the synaptic cytosol and the enriched PSD fraction. The protocol can also potentially be adapted to other hiPSC-derived neuronal types, with necessary changes made to cell seeding density and buffer volumes.

Droplet-based transcriptome profiling of individual synapses.

Synapses are crucial structures that mediate signal transmission between neurons in complex neural circuits and display considerable morphological and electrophysiological heterogeneity. So far we still lack a high-throughput method to profile the molecular heterogeneity among individual synapses. In the present study, we develop a droplet-based single-cell (sc) total-RNA-sequencing platform, called Multiple-Annealing-and-Tailing-based Quantitative scRNA-seq in Droplets, for transcriptome profiling of individual neurites, primarily composed of synaptosomes. In the synaptosome transcriptome, or 'synaptome', profiling of both mouse and human brain samples, we detect subclusters among synaptosomes that are associated with neuronal subtypes and characterize the landscape of transcript splicing that occurs within synapses. We extend synaptome profiling to synaptopathy in an Alzheimer's disease (AD) mouse model and discover AD-associated synaptic gene expression changes that cannot be detected by single-nucleus transcriptome profiling. Overall, our results show that this platform provides a high-throughput, single-synaptosome transcriptome profiling tool that will facilitate future discoveries in neuroscience.

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.

Alzheimer's disease-associated R47H TREM2 increases, but wild-type TREM2 decreases, microglial phagocytosis of synaptosomes and neuronal loss.

Triggering receptor on myeloid cells 2 (TREM2) is an innate immune receptor, upregulated on the surface of microglia associated with amyloid plaques in Alzheimer's disease (AD). Individuals heterozygous for the R47H variant of TREM2 have greatly increased risk of developing AD. We examined the effects of wild-type (WT), R47H and knock-out (KO) of human TREM2 expression in three microglial cell systems. Addition of mouse BV-2 microglia expressing R47H TREM2 to primary mouse neuronal cultures caused neuronal loss, not observed with WT TREM2. Neuronal loss was prevented by using annexin V to block exposed phosphatidylserine, an eat-me signal and ligand of TREM2, suggesting loss was mediated by microglial phagocytosis of neurons exposing phosphatidylserine. Addition of human CHME-3 microglia expressing R47H TREM2 to LUHMES neuronal-like cells also caused loss compared to WT TREM2. Expression of R47H TREM2 in BV-2 and CHME-3 microglia increased their uptake of phosphatidylserine-beads and synaptosomes versus WT TREM2. Human iPSC-derived microglia with heterozygous R47H TREM2 had increased phagocytosis of synaptosomes vs common-variant TREM2. Additionally, phosphatidylserine liposomes increased activation of human iPSC-derived microglia expressing homozygous R47H TREM2 versus common-variant TREM2. Finally, overexpression of TREM2 in CHME-3 microglia caused increased expression of cystatin F, a cysteine protease inhibitor, and knock-down of cystatin F increased CHME-3 uptake of phosphatidylserine-beads. Together, these data suggest that R47H TREM2 may increase AD risk by increasing phagocytosis of synapses and neurons via greater activation by phosphatidylserine and that WT TREM2 may decrease microglial phagocytosis of synapses and neurons via cystatin F.

Central nervous system specific high molecular weight ALS2/alsin homophilic complex is enriched in mouse brain synaptosomes.

ALS2/alsin, the causative gene product for a number of juvenile recessive motor neuron diseases, acts as a guanine nucleotide exchange factor (GEF) for Rab5, regulating early endosome trafficking and maturation. It has been demonstrated that ALS2 forms a tetramer, and this oligomerization is essential for its GEF activity and endosomal localization in established cancer cells. However, despite that ALS2 deficiency is implicated in neurological diseases, neither the subcellular distribution of ALS2 nor the form of its complex in the central nervous system (CNS) has been investigated. In this study, we showed that ALS2 in the brain was enriched both in synaptosomal and cytosolic fractions, while those in the liver were almost exclusively present in cytosolic fraction by differential centrifugation. Gel filtration chromatography revealed that cytosolic ALS2 prepared both from the brain and liver formed a tetramer. Remarkably, synaptosomal ALS2 existed as a high-molecular weight complex in addition to a tetramer. Such complex was also observed not only in embryonic brain but also several neuronal and glial cultures, but not in fibroblast-derived cell lines. Thus, the high-molecular weight ALS2 complex represents a unique form of ALS2-homophilic oligomers in the CNS, which may play a role in the maintenance of neural function.

Early time course of oxidative stress in hippocampal synaptosomes and cognitive loss following impaired insulin signaling in rats: Development of sporadic Alzheimer's disease.

Oxidative stress, caused by impaired insulin signaling, plays a pivotal role in the pathogenesis of sporadic Alzheimer's disease (sAD). We investigated the oxidative stress parameters in the synaptosomes prepared from the hippocampus tissue in order to identify their potential role in sAD development in intraperitoneal (IP) and intracerebroventricular (ICV) streptozotocin (STZ) injections models of insulin signaling impairment. Rats were harvested 1, 3, or 6 weeks post treatment. Spatial learning and memory, several antioxidants and oxidative stress markers were analyzed. Results showed a significant deficit in learning and memory in rats injected with STZ through IP and ICV routes. Glutathione, glutathione/oxidized glutathione, glutathione S-transferase, glutathione peroxidase, glutathione reductase, catalase, superoxide dismutase(SOD)-total, Zn/Cu(SOD), Mn/Fe(SOD) are significantly decreased in IP-STZ and ICV-STZ groups at 1, 3, and 6 weeks after STZ injection. Oxidized glutathione, thiobarbituric acid reactive species, glucose 6-Phosphate dehydrogenase, protein carbonyls, 4-Hydroxynonenal, and 3-Nitrotyrosine are significantly increased in IP-STZ and ICV-STZ groups at 1,3, and 6 weeks after STZ injection. Changes in oxidative stress parameters in ICV-STZ groups are greater than IP-STZ groups. STZ treatment induced cognitive impairments by 3-W and 6-W, and it was significantly correlated with the extent of oxidative damage. In conclusion, STZ administration through ICV route is deleterious in causing early synaptosomal oxidative damage that exacerbated with time and correlated with cognitive impairments. Our data implicate the involvement of oxidative stress as an early feature of sAD and provide insights into the behavioral and biochemical changes over the course of disease development.

Inhibition of Synaptic Glutamate Exocytosis and Prevention of Glutamate Neurotoxicity by Eupatilin from Artemisia argyi in the Rat Cortex.

The inhibition of synaptic glutamate release to maintain glutamate homeostasis contributes to the alleviation of neuronal cell injury, and accumulating evidence suggests that natural products can repress glutamate levels and associated excitotoxicity. In this study, we investigated whether eupatilin, a constituent of Artemisia argyi, affected glutamate release in rat cortical nerve terminals (synaptosomes). Additionally, we evaluated the effect of eupatilin in an animal model of kainic acid (KA) excitotoxicity, particularly on the levels of glutamate and N-methyl-D-aspartate (NMDA) receptor subunits (GluN2A and GluN2B). We found that eupatilin decreased depolarization-evoked glutamate release from rat cortical synaptosomes and that this effect was accompanied by a reduction in cytosolic Ca2+ elevation, inhibition of P/Q-type Ca2+ channels, decreased synapsin I Ca2+-dependent phosphorylation and no detectable effect on the membrane potential. In a KA-induced glutamate excitotoxicity rat model, the administration of eupatilin before KA administration prevented neuronal cell degeneration, glutamate elevation, glutamate-generating enzyme glutaminase increase, excitatory amino acid transporter (EAAT) decrease, GluN2A protein decrease and GluN2B protein increase in the rat cortex. Taken together, the results suggest that eupatilin depresses glutamate exocytosis from cerebrocortical synaptosomes by decreasing P/Q-type Ca2+ channels and synapsin I phosphorylation and alleviates glutamate excitotoxicity caused by KA by preventing glutamatergic alterations in the rat cortex. Thus, this study suggests that eupatilin can be considered a potential therapeutic agent in the treatment of brain impairment associated with glutamate excitotoxicity.

Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain.

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological disorders. The characterization of synaptic sub-compartments by biochemical isolation is, therefore, a powerful method to elucidate the molecular bases of synaptic processes, both in health and disease. This protocol describes the isolation of synaptic terminals and synaptic sub-compartments from mouse brains by subcellular fractionation. First, sealed synaptic terminal structures, known as synaptosomes, are isolated following brain tissue homogenization. Synaptosomes are neuronal pre- and post-synaptic compartments with pinched-off and sealed membranes. These structures retain a metabolically active state and are valuable for studying synaptic structure and function. The synaptosomes are then subjected to hypotonic lysis and ultracentrifugation to obtain synaptic sub-compartments enriched for synaptic vesicles, synaptic cytosol, and synaptic plasma membrane. Fraction purity is confirmed by electron microscopy and biochemical enrichment analysis for proteins specific to sub-synaptic compartments. The presented method is a straightforward and valuable tool for studying the structural and functional characteristics of the synapse and the molecular etiology of various brain disorders.