Search

Amyloid β (Aβ) peptides accumulating in the brain are proposed to trigger Alzheimer's disease (AD). However, molecular cascades underlying their toxicity are poorly defined. Here, we explored a novel hypothesis for Aβ42 toxicity that arises from its proven affinity for γ-secretases. We hypothesized that the reported increases in Aβ42, particularly in the endolysosomal compartment, promote the establishment of a product feedback inhibitory mechanism on γ-secretases, and thereby impair downstream signaling events. We conducted kinetic analyses of γ-secretase activity in cell-free systems in the presence of Aβ, as well as cell-based and ex vivo assays in neuronal cell lines, neurons, and brain synaptosomes to assess the impact of Aβ on γ-secretases. We show that human Aβ42 peptides, but neither murine Aβ42 nor human Aβ17-42 (p3), inhibit γ-secretases and trigger accumulation of unprocessed substrates in neurons, including C-terminal fragments (CTFs) of APP, p75, and pan-cadherin. Moreover, Aβ42 treatment dysregulated cellular homeostasis, as shown by the induction of p75-dependent neuronal death in two distinct cellular systems. Our findings raise the possibility that pathological elevations in Aβ42 contribute to cellular toxicity via the γ-secretase inhibition, and provide a novel conceptual framework to address Aβ toxicity in the context of γ-secretase-dependent homeostatic signaling. [ABSTRACT FROM AUTHOR]

Cystatin B (CSTB) is a small protease inhibitor protein being involved in cell proliferation and neuronal differentiation. Loss-of-function mutations in CSTB gene cause progressive myoclonic epilepsy 1 (EPM1). We previously demonstrated that CSTB is locally synthesized in synaptic nerve terminals from rat brain and secreted into the media, indicating its role in synaptic plasticity. In this work, we have further investigated the involvement of CSTB in synaptic plasticity, using synaptosomes from human cerebral organoids (hCOs) as well as from rodents' brain. Our data demonstrate that CSTB is released from synaptosomes in two ways: (i) as a soluble protein and (ii) in extracellular vesicles-mediated pathway. Synaptosomes isolated from hCOs are enriched in pre-synaptic proteins and contain CSTB at all developmental stages analyzed. CSTB presence in the synaptic territories was also confirmed by immunostaining on human neurons in vitro. To investigate if the depletion of CSTB affects synaptic plasticity, we characterized the synaptosomes from EPM1 hCOs. We found that the levels of presynaptic proteins and of an initiation factor linked to local protein synthesis were both reduced in EPM1 hCOs and that the extracellular vesicles trafficking pathway was impaired. Moreover, EPM1 neurons displayed anomalous morphology with longer and more branched neurites bearing higher number of intersections and nodes, suggesting connectivity alterations. In conclusion, our data strengthen the idea that CSTB plays a critical role in the synapse physiology and reveal that pathologically low levels of CSTB may affect synaptic plasticity, leading to synaptopathy and altered neuronal morphology. [ABSTRACT FROM AUTHOR]

Background and Purpose: To deepen our knowledge of the role of complement in synaptic impairment in experimental autoimmune encephalomyelitis (EAE) mice, we investigated the distribution of C1q and C3 proteins and the role of complement as a promoter of glutamate release in purified nerve endings (synaptosomes) and astrocytic processes (gliosomes) isolated from the cortex of EAE mice at the acute stage of the disease (21 ± 1 day post‐immunization). Experimental Approach: EAE cortical synaptosomes and gliosomes were analysed for glutamate release efficiency (measured as release of preloaded [3H]D‐aspartate ([3H]D‐ASP)), C1q and C3 protein density, and for viability and ongoing apoptosis. Key Results: In healthy mice, complement releases [3H]D‐ASP from gliosomes more efficiently than from synaptosomes. The releasing activity occurs in a dilution‐dependent manner and involves the reversal of the excitatory amino acid transporters (EAATs). In EAE mice, the complement‐induced releasing activity is significantly reduced in cortical synaptosomes but amplified in cortical gliosomes. These adaptations are paralleled by decreased density of the EAAT2 protein in synaptosomes and increased EAAT1 staining in gliosomes. Concomitantly, PSD95, GFAP, and CD11b, but not SNAP25, proteins are overexpressed in the cortex of the EAE mice. Similarly, C1q and C3 protein immunostaining is increased in EAE cortical synaptosomes and gliosomes, although signs of ongoing apoptosis or altered viability are not detectable. Conclusion and Implications: Our results unveil a new noncanonical role of complement in the CNS of EAE mice relevant to disease progression and central synaptopathy that suggests new therapeutic targets for the management of MS. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a severe neurodegenerative condition affecting millions worldwide. Prevalence of AD correlates with increased life expectancy and aging population in the developed countries. Considering that AD is a multifactorial disease involving various pathological processes such as synaptic dysfunction, neuroinflammation, oxidative stress, and improper protein folding, a comprehensive approach targeting multiple pathways may prove effective in slowing the disease progression. Cellular therapy and its further development in the form of cell vesicle and particularly mitochondrial transplantation represent promising approaches for treating neurodegeneration. The use of synaptosomes, due to uniqueness of their contents, could mark a new stage in the development of comprehensive therapies for neurodegenerative diseases, particularly AD. Synaptosomes contain unique memory mitochondria, which differ not only in size but also in functionality compared to the mitochondria in the neuronal soma. These synaptosomal mitochondria actively participate in cellular communication and signal transmission within synapses. Synaptosomes also contain other elements such as their own protein synthesis machinery, synaptic vesicles with neurotransmitters, synaptic adhesion molecules, and microRNAs – all crucial for synaptic transmission and, consequently, cognitive processes. Complex molecular ensemble ensures maintenance of the synaptic autonomy of mitochondria. Additionally, synaptosomes, with their affinity for neurons, can serve as an optimal platform for targeted drug delivery to nerve cells. This review discusses unique composition of synaptosomes, their capabilities and advantages, as well as limitations of their suggested use as therapeutic agents for treating neurodegenerative pathologies, particularly AD. [ABSTRACT FROM AUTHOR]

The adenosine modulation system is mostly composed by inhibitory A1 receptors (A1R) and the less abundant facilitatory A2A receptors (A2AR), the latter selectively engaged at high frequency stimulation associated with synaptic plasticity processes in the hippocampus. A2AR are activated by adenosine originated from extracellular ATP through ecto-5'-nucleotidase or CD73-mediated catabolism. Using hippocampal synaptosomes, we now investigated how adenosine receptors modulate the synaptic release of ATP. The A2AR agonist CGS21680 (10-100 nM) enhanced the K+-evoked release of ATP, whereas both SCH58261 and the CD73 inhibitor α,β-methylene ADP (100 μM) decreased ATP release; all these effects were abolished in forebrain A2AR knockout mice. The A1R agonist CPA (10-100 nM) inhibited ATP release, whereas the A1R antagonist DPCPX (100 nM) was devoid of effects. The presence of SCH58261 potentiated CPA-mediated ATP release and uncovered a facilitatory effect of DPCPX. Overall, these findings indicate that ATP release is predominantly controlled by A2AR, which are involved in an apparent feedback loop of A2AR-mediated increased ATP release together with dampening of A1R-mediated inhibition. This study is a tribute to María Teresa Miras-Portugal. [ABSTRACT FROM AUTHOR]

Recent evidence suggests that ferroptosis, an iron‐dependent cell death process, may be involved in Alzheimer's disease (AD) pathology. The study evaluated the therapeutic potential of betaine and boric acid (BA) pretreatment administered to rats for 21 days in AD. Then, the rats were sacrificed, and morphological and biochemical analyses were performed in brain tissues. Next, an ex vivo AD model was created by applying amyloid‐β (Aβ1‐42) to synaptosomes isolated from the brain tissues. Synaptosomes were analyzed with micrograph images, and protein and mRNA levels of ferroptotic markers were determined. Betaine and BA pretreatments did not cause any morphological and biochemical differences in the brain tissue. However, Aβ (1–42) administration in synaptosomes increased the levels of acyl‐CoA synthetase long chain family member‐4 (ACSL4), transferrin receptor‐1 protein (TfR1), malondialdehyde (MDA), and 8‐hydroxydeoxyguanosine (8‐OHdG) and decreased the levels glutathione peroxidase‐4 (GPx4) and glutathione (GSH). Moreover, ACSL4, GPx4, and TfR1 mRNA and protein levels were similar to the ELISA results. In contrast, betaine and BA pretreatments decreased the levels of ACSL4, TfR1, MDA, and 8‐OHdG in synaptosomes incubated with Aβ1‐42, while promoting increased levels of GPx4 and GSH. In addition, betaine and BA pretreatments completely reversed ACSL4, GPx4, and TfR1 mRNA and protein levels. Therefore, betaine and BA pretreatments may contribute to the prevention of neurodegenerative damage by supporting antiferroptotic activities. [ABSTRACT FROM AUTHOR]

INTRODUCTION: Early‐life stress (ES) increases the risk for Alzheimer's disease (AD). We and others have shown that ES aggravates amyloid‐beta (Aβ) pathology and promotes cognitive dysfunction in APP/PS1 mice, but underlying mechanisms remain unclear. METHODS: We studied how ES affects the hippocampal synaptic proteome in wild‐type (WT) and APP/PS1 mice at early and late pathological stages, and validated hits using electron microscopy and immunofluorescence. RESULTS: The hippocampal synaptosomes of both ES‐exposed WT and early‐stage APP/PS1 mice showed a relative decrease in actin dynamics‐related proteins and a relative increase in mitochondrial proteins. ES had minimal effects on older WT mice, while strongly affecting the synaptic proteome of advanced stage APP/PS1 mice, particularly the expression of astrocytic and mitochondrial proteins. DISCUSSION: Our data show that ES and amyloidosis share pathogenic pathways involving synaptic mitochondrial dysfunction and lipid metabolism, which may underlie the observed impact of ES on the trajectory of AD. [ABSTRACT FROM AUTHOR]

Glutamate is a major excitatory neurotransmitter that mediates neuronal damage in acute and chronic brain disorders. The effect and mechanism of phillygenin, a natural compound with neuroprotective potential, on glutamate release in isolated nerve terminals (synaptosomes) prepared from the rat cerebral cortex were examined. In this study, 4-aminopyridine (4-AP), a potassium channel blocker, was utilized to induce the release of glutamate, which was subsequently quantified via a fluorometric assay. Our findings revealed that phillygenin reduced 4-AP-induced glutamate release, and this inhibitory effect was reversed by removing extracellular Ca2+ or inhibiting vesicular transport with bafilomycin A1. However, exposure to the glutamate transporter inhibitor dl-threo-beta-benzyl-oxyaspartate (dl-TOBA) did not influence the inhibitory effect. Moreover, phillygenin did not change the synaptosomal membrane potential but lowered the 4-AP-triggered increase in intrasynaptosomal Ca2+ concentration ([Ca2+]i). Antagonizing Cav2.2 (N-type) calcium channels blocked the inhibition of glutamate release by phillygenin, whereas pretreatment with the mitochondrial Na+/Ca2+ exchanger inhibitor, CGP37157 or the ryanodine receptor inhibitor, dantrolene, both of which block intracellular Ca2+ release, had no effect. The effect of phillygenin on glutamate release triggered by 4-AP was completely abolished when MAPK/ERK inhibitors were applied. Furthermore, phillygenin attenuated the phosphorylation of ERK1/2 and its major presynaptic target, synapsin I, a protein associated with synaptic vesicles. These data collectively suggest that phillygenin mediates the inhibition of evoked glutamate release from synaptosomes primarily by reducing the influx of Ca2+ through Cav2.2 calcium channels, thereby subsequently suppressing the MAPK/ERK/synapsin I signaling cascade. [ABSTRACT FROM AUTHOR]

Sphingosine-1-phosphate (S1P) is a phosphosphingolipid with pleiotropic biological functions. S1P acts as an intracellular second messenger, as well as extracellular ligand to five G-protein coupled receptors (S1PR1-5). In the brain, S1P regulates neuronal proliferation, apoptosis, synaptic activity and neuroglia activation. Moreover, S1P metabolism alterations have been reported in neurodegenerative disorders. We have previously reported that S1PRs are present in nerve terminals, exhibiting distinct sub-synaptic localization and neuromodulation actions. Since type 2 diabetes (T2D) causes synaptic dysfunction, we hypothesized that S1P signaling is modified in nerve terminals. In this study, we determined the density of S1PRs in cortical synaptosomes from insulin-resistant Goto-Kakizaki (GK) rats and Wistar controls, and from mice fed a high-fat diet (HFD) and low-fat-fed controls. Relative to their controls, GK rats showed similar cortical S1P concentration despite higher S1P levels in plasma, yet lower density of S1PR1, S1PR2 and S1PR4 in nerve-terminal-enriched membranes. HFD-fed mice exhibited increased plasma and cortical concentrations of S1P, and decreased density of S1PR1 and S1PR4. These findings point towards altered S1P signaling in synapses of insulin resistance and diet-induced obesity models, suggesting a role of S1P signaling in T2D-associated synaptic dysfunction. [ABSTRACT FROM AUTHOR]

Aim. Xenobiotic metal mercury is one of the major crucial pollutants of global public health concerns according to the World Health Organization assessment. Carbon-containing nanoparticles (CNPs) are promising in nanotechnology. CNPs were obtained by the combustion of citric acid and urea. Methods. The cortex nerve terminals isolated from Wistar rats were used in the experiments. [14C]glutamate uptake and release in the nerve terminals were monitored using a radiolabeled assay. Results. It was shown that HgCl2 starting from 5 µM caused a concentration-dependent increase in the extracellular L-[14C]glutamate level in nerve terminals resulted from weak functioning of glutamate transporter, and so significantly decreased L-[14C] glutamate uptake. Combined effects of Hg2+ and CNPs obtained by heating of citric acid and urea were analysed. CNPs were able to mitigate in an acute manner excitotoxic Hg2+-induced increase in the extracellular L-[14C]glutamate level in nerve terminals by 37%, thereby being a provisional Hg2+ scavenger. Conclusion. Besides biotechnological implementation of data, developed approach can be applicable for monitoring capability of different particles and compounds to mitigate Hg2+ -mediated threat.

Synapses are the cellular substrates of higher-order brain functions, and their dysfunction is an early and primary pathogenic mechanism across several neurological disorders. In particular, Alzheimer's disease (AD) is categorized by prodromal structural and functional synaptic deficits, prior to the advent of classical behavioral and pathological features. Recent research has shown that the development, maintenance, and plasticity of synapses depend on localized protein translation. Synaptosomes and synaptoneurosomes are biochemically isolated synaptic terminal preparations which have long been used to examine a variety of synaptic processes ex vivo in both healthy and pathological conditions. These ex vivo preparations preserve the mRNA species and the protein translational machinery. Hence, they are excellent in organello tools for the study of alterations in mRNA levels and protein translation in neuropathologies. Evaluation of synapse-specific basal and activity-driven de novo protein translation activity can be conveniently performed in synaptosomal/synaptoneurosomal preparations from both rodent and human brain tissue samples. This review gives a quick overview of the methods for isolating synaptosomes and synaptoneurosomes before discussing the studies that have utilized these preparations to study localized synapse-specific protein translation in (patho)physiological situations, with an emphasis on AD. While the review is not an exhaustive accumulation of all the studies evaluating synaptic protein translation using the synaptosomal model, the aim is to assemble the most relevant studies that have done so. The hope is to provide a suitable research platform to aid neuroscientists to utilize the synaptosomal/synaptoneurosomal models to evaluate the molecular mechanisms of synaptic dysfunction within the specific confines of mRNA localization and protein translation research. [ABSTRACT FROM AUTHOR]

Recent findings suggest an important role for the dysregulation of stromal interaction molecule (STIM) proteins, activators of store-operated Ca2+ channels, and the prolonged activation of N-methyl-D-aspartate receptors (NMDARs) in the development of neurodegenerative diseases. We previously demonstrated that STIM silencing increases Ca2+ influx through NMDAR and STIM–NMDAR2 complexes are present in neurons. However, the interplay between NMDAR subunits (GluN1, GluN2A, and GluN2B) and STIM1/STIM2 with regard to intracellular trafficking remains unknown. Here, we found that the activation of NMDAR endocytosis led to an increase in STIM2–GluN2A and STIM2–GluN2B interactions in primary cortical neurons. STIM1 appeared to migrate from synaptic to extrasynaptic sites. STIM2 silencing inhibited post-activation NMDAR translocation from the plasma membrane and synaptic spines and increased NMDAR currents. Our findings reveal a novel molecular mechanism by which STIM2 regulates NMDAR synaptic trafficking by promoting NMDAR endocytosis after receptor overactivation, which may suggest protection against excessive uncontrolled Ca2+ influx through NMDARs. [ABSTRACT FROM AUTHOR]

Some of the prominent features of long-term memory formation include protein synthesis, gene expression, enhanced neurotransmitter release, increased excitability, and formation of new synapses. As these processes are critically dependent on mitochondrial function, we hypothesized that increased mitochondrial respiration and dynamics would play a prominent role in memory formation. To address this possibility, we measured mitochondrial oxygen consumption (OCR) in hippocampal tissue punches from trained and untrained animals. Our results show that context fear training significantly increased basal, ATP synthesis-linked, and maximal OCR in the Shaffer collateral-CA1 synaptic region, but not in the CA1 cell body layer. These changes were recapitulated in synaptosomes isolated from the hippocampi of fear-trained animals. As dynamin-related protein 1 (Drp1) plays an important role in mitochondrial fission, we examined its role in the increased mitochondrial respiration observed after fear training. Drp1 inhibitors decreased the training-associated enhancement of OCR and impaired contextual fear memory, but did not alter the number of synaptosomes containing mitochondria. Taken together, our results show context fear training increases presynaptic mitochondria respiration, and that Drp-1 mediated enhanced energy production in CA1 pre-synaptic terminals is necessary for context fear memory that does not result from an increase in the number of synaptosomes containing mitochondria or an increase in mitochondrial mass within the synaptic layer. [ABSTRACT FROM AUTHOR]

• A synaptosomal preparation enriched in functional intrasynaptic mitochondria. • An isolation protocol for the obtention of intact synaptosomes is described. • Synaptosomal integrity and specificity were confirmed by calcein and SNAP-25 assays. • Intrasynaptic mitochondria show adequate structural and functional integrity. 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. [ABSTRACT FROM AUTHOR]

Abstract Background The amyloid precursor protein (APP), a key player in Alzheimer’s disease (AD), is part of a larger gene family, including the APP like proteins APLP1 and APLP2. They share similar structures, form homo- and heterotypic dimers and exhibit overlapping functions. Results We investigated complex formation of the APP family members via two inducible dimerization systems, the FKBP-rapamycin based dimerization as well as cysteine induced dimerization, combined with co-immunoprecipitations and Blue Native (BN) gel analyses. Within the APP family, APLP1 shows the highest degree of dimerization and high molecular weight (HMW) complex formation. Interestingly, only about 20% of APP is dimerized in cultured cells whereas up to 50% of APP is dimerized in mouse brains, independent of age and splice forms. Furthermore, we could show that dimerized APP originates mostly from neurons and is enriched in synaptosomes. Finally, BN gel analysis of human cortex samples shows a significant decrease of APP dimers in AD patients compared to controls. Conclusions Together, we suggest that loss of full-length APP dimers might correlate with loss of synapses in the process of AD.

Synaptic transfer of tau has long been hypothesized from the human pathology pattern and has been demonstrated in vitro and in vivo, but the precise mechanisms remain unclear. Extracellular vesicles such as exosomes have been suggested as a mechanism, but not all tau is exosomal. The present experiments use a novel flow cytometry assay to quantify depolarization of synaptosomes by KCl after loading with FM2-10, which induces a fluorescence reduction associated with synaptic vesicle release; the degree of reduction in cryopreserved human samples equaled that seen in fresh mouse synaptosomes. Depolarization induced the release of vesicles in the size range of exosomes, along with tetraspanin markers of extracellular vesicles. A number of tau peptides were released, including tau oligomers; released tau was primarily unphosphorylated and C-terminal truncated, with Aβ release just above background. When exosomes were immunopurified from release supernatants, a prominent tau band showed a dark smeared appearance of SDS-stable oligomers along with the exosomal marker syntenin-1, and these exosomes induced aggregation in the HEK tau biosensor assay. However, the flow-through did not seed aggregation. Size exclusion chromatography of purified released exosomes shows faint signals from tau in the same fractions that show a CD63 band, an exosomal size signal, and seeding activity. Crude synaptosomes from control, tauopathy, and AD cases demonstrated lower seeding in tauopathy compared to AD that is correlated with the measured Aβ42 level. These results show that AD synapses release exosomal tau that is C-terminal-truncated, oligomeric, and with seeding activity that is enhanced by Aβ. Taken together with previous findings, these results are consistent with a direct prion-like heterotypic seeding of tau by Aβ within synaptic terminals, with subsequent loading of aggregated tau onto exosomes that are released and competent for tau seeding activity.

Synaptic refinement is a critical physiological process that removes excess synapses to establish and maintain functional neuronal circuits. Recent studies have shown that focal exposure of phosphatidylserine (PS) on synapses acts as an "eat me" signal to mediate synaptic pruning. However, the molecular mechanism underlying PS externalization at synapses remains elusive. Here, we find that murine CDC50A, a chaperone of phospholipid flippases, localizes to synapses, and that its expression depends on neuronal activity. Cdc50a knockdown leads to phosphatidylserine exposure at synapses and subsequent erroneous synapse removal by microglia partly via the GPR56 pathway. Taken together, our data support that CDC50A safeguards synapse maintenance by regulating focal phosphatidylserine exposure at synapses.

The aim of this study was to analyse a capability of carbon-containing nanoparticles (CNPs) obtained by heating of organics, to influence mercury-induced neurotoxicity in biological system, such as presynaptic rat cortex nerve terminals. Methods. CNPs were obtained using method described in [6] by the combustion of citric acid and urea. The cortex nerve terminals isolated from Wistar rats were used in the experiments. [14C] glutamate uptake and release in the nerve terminals were monitored using a radiolabelled assay. In particular, rat brain nerve terminals (synaptosomes) were isolated from the rat cortex. The cortex regions were rapidly removed and homogenized in the ice-cold solution consisted of: sucrose 0.32 M; HEPES-NaOH 5 mM, pH 7.4; EDTA 0.2 mM. One synaptosomal preparation was isolated from one rat. The synaptosomes from brain homogenate were obtained according to the procedure proposed by Cotman with minor modifications [7] by differential centrifugation and Ficoll-400 density gradient centrifugation. The concentrations of proteins were monitored according to Larson. To measure the uptake of L-[14C] glutamate, the synaptosomal suspension was pre-incubated in the standard saline solution. Then, HgCl2 was applied to the synaptosomal incubation media, and synaptosomes were further incubated for 6 min before starting the uptake, which in turn was initiated by the application of the aliquots of non-radiolabelled L-glutamate (10 μM) supplemented with L-[14C] glutamate, 420 nM, 0.1 μCi/ml, and then the synaptosomes were incubated at 37 °C during 1 min to measure the initial rate of L-[14C] glutamate uptake. L-[14C] glutamate uptake was monitored with liquid scintillation counting using the ACS scintillation cocktail, 1.5 ml [8]. To measure the extracellular level of L-[14C] glutamate, the synaptosomes were were preincubated at 37 °C during 10 min to restore the ion gradients, and after that they were loaded with L-[14C] glutamate, 1 nmol per mg of protein, 238 mCi/mmol, in the standard saline solution at 37 °C during 10 min according to [9]. Total synaptosomal content of L-[14C] glutamate was equal to 200000±15000 cpm/mg protein. Results. In the first sets of the experiments, Hg2+ effects on the extracellular level of L-[14C] glutamate were assessed in nerve terminal preparations (Fig.1). It was shown a mercury-induced excitotoxic increase in the ambient level of L-[14C] glutamate in nerve terminal preparations. In the second sets of the experiments (Fig. 2), it was demonstrated that Hg2+ decreased the initial rate and accumulation of L-[14C] glutamate by nerve terminals starting from a concentration of 10 μM. Therefore, it was shown that a mercury-induced excitotoxic increase in the ambient level of L-[14C] glutamate in nerve terminal preparations (Fig. 1) resulted from weak functioning of glutamate transporter, and so significantly decreased L-[14C] glutamate uptake (Fig. 2). In the third sets of the experiments, it was shown that CNPs from heating of citric acid/urea mitigated an excitotoxic mercury-induced increase in the extracellular level of L-[14C] glutamate in nerve terminal preparations. The latter was equal to 0.425 ± 0.023 nmol/mg of proteins after combined application of HgCl2 (5 μM) and CNPs (1 mg/ml) (Р ≤ 0.05 as compared to effect of Hg2+per se; n = 6) and 0.460 ± 0.017 nmol/mg of proteins after combined application of HgCl2 (5 μM) and CNPs (10 mg/ml) (Р ≤ 0.05 as compared to effect of Hg2+ per se; n = 6). Therefore, CNPs were able to mitigate in an acute manner excitotoxic Hg2+-induced increase in the extracellular L-[14C]glutamate level in nerve terminals by 37%, thereby being a provisional Hg2+ scavenger. Conclusions. CNPs can mitigate Hg2+-induced excitotoxicity in nerve terminals. Taking into account this fact, it can be assumed that these nanoparticles can be used as Hg2+ adsorbent in the human organism. Besides biotechnological implementation of data, developed approach can be applicable for monitoring capability of different particles and compounds to mitigate Hg2+-mediated threat. [ABSTRACT FROM AUTHOR]

Traumatic brain injury (TBI) causes significant neurological deficits and long‐term degenerative changes. Primary injury in TBI entails distinct neuroanatomical zones, i.e., contusion (Ct) and pericontusion (PC). Their dynamic expansion could contribute to unpredictable neurological deterioration in patients. Molecular characterization of these zones compared with away from contusion (AC) zone is invaluable for TBI management. Using proteomics‐based approach, we were able to distinguish Ct, PC and AC zones in human TBI brains. Ct was associated with structural changes (blood–brain barrier (BBB) disruption, neuroinflammation, axonal injury, demyelination and ferroptosis), while PC was associated with initial events of secondary injury (glutamate excitotoxicity, glial activation, accumulation of cytoskeleton proteins, oxidative stress, endocytosis) and AC displayed mitochondrial dysfunction that could contribute to secondary injury events and trigger long‐term degenerative changes. Phosphoproteome analysis in these zones revealed that certain differentially phosphorylated proteins synergistically contribute to the injury events along with the differentially expressed proteins. Non‐synaptic mitochondria (ns‐mito) was associated with relatively more differentially expressed proteins (DEPs) compared to synaptosomes (Syn), while the latter displayed increased protein oxidation including tryptophan (Trp) oxidation. Proteomic analysis of immunocaptured complex I (CI) from Syn revealed increased Trp oxidation in Ct > PC > AC (vs. control). Oxidized W272 in the ND1 subunit of CI, revealed local conformational changes in ND1 and the neighboring subunits, as indicated by molecular dynamics simulation (MDS). Taken together, neuroanatomical zones in TBI show distinct protein profile and protein oxidation representing different primary and secondary injury events with potential implications for TBI pathology and neurological status of the patients. [ABSTRACT FROM AUTHOR]

Microglia have been implicated in the pathophysiology of major depressive disorder (MDD), but information on biological mechanisms is limited. Therefore, we investigated the gene expression profile of microglial cells in relation to neuronal regulators of microglia activity in well-characterized MDD and control autopsy brains. Pure, intact microglia were isolated at brain autopsy from occipital cortex gray matter (GM) and corpus callosum white matter of 13 donors with MDD and 10 age-matched control donors for RNA sequencing. Top differentially expressed genes were validated using immunohistochemistry staining. Because gene expression changes were only detected in GM microglia, neuronal regulators of microglia were investigated in cortical tissue and synaptosomes from the cortex by reverse transcriptase–quantitative polymerase chain reaction and Western blot. Transcriptome analysis revealed 92 genes differentially expressed in microglia isolated from GM, but none in microglia from white matter in donors with MDD, compared with control donors. Of these, 81 genes were less abundantly expressed in GM in MDD, including CD163 , MKI67 , SPP1 , CD14 , FCGR1A / C , and C1QA / B / C. Accordingly, pathways related to effector mechanisms, such as the complement system and phagocytosis, were differentially regulated in GM microglia in MDD. Immunohistochemistry staining revealed significantly lower expression of CD163 protein in MDD. Whole tissue analysis showed an increase in CD200 (p =.0009) and CD47 (p =.068) messenger RNA, and CD47 protein was significantly elevated (p =.0396) in synaptic fractions of MDD cases. Transcriptional profiling indicates an immune-suppressed microglial phenotype in MDD that is possibly caused by neuronal regulation. [ABSTRACT FROM AUTHOR]

A comprehensive review was conducted to compile the contributions of Mary B. Dratman and studies by other researchers in the field of nongenomic actions of thyroid hormones in adult mammalian brain. Dratman and her collaborators authored roughly half of the papers in this area. It has been almost fifty years since Dratman introduced the novel concept of thyroid hormones as neurotransmitters for the first time. The characterization of unique brainregion specific accumulation of thyroid hormones within the nerve terminals in adult mammals was a remarkable contribution by Dratman. It suggested a neurotransmitter- or neuromodulator-like role of thyroid hormone and/or its derivative, 3-iodothyronamine within adrenergic systems in adult mammalian brain. Several studies by other researchers using synaptosomes as a model system, have contributed to the concept of direct nongenomic actions of thyroid hormones at synaptic regions by establishing that thyroid hormones or their derivatives can bind to synaptosomal membranes, alter membrane functions including enzymatic activities and ion transport, elicit Ca2+/NOdependent signaling pathways and induce substrate-protein phosphorylation. Such findings can help to explain the physiological and pathophysiological roles of thyroid hormone in psychobehavioral control in adult mammalian brain. However, the exact mode of nongenomic actions of thyroid hormones at nerve terminals in adult mammalian brain awaits further study. [ABSTRACT FROM AUTHOR]

This study provides evidence of the existence of presynaptic inhibitory sphingosine-1-phosphate receptor 1 (S1P1R) and facilitatory S1P3R in cortical nerve endings (synaptosomes) of healthy mice. The conclusion relies on the findings that (i) the S1P1R agonist CS-2100 (0.1–30 nM) inhibits the 12 mM KCl-evoked glutamate exocytosis (quantified as the release of [3H]D-aspartate) while the S1P3R allosteric agonist CYM-5541 potentiates it and (ii) these effects are inhibited by the S1P1R antagonist Ex 26 (30–300 nM) and the S1P3R antagonist TY-52156 (100–1000 nM), respectively. Confocal microscopy and western blot analysis confirmed the presence of S1P1R and S1P3R proteins in cortical glutamatergic synaptosomes, which were scarcely accessible to biotin in a biotinylation study. Then, we demonstrated that S1P1R and S1P3R densities and their release activity are amplified in cortical synaptosomes of mice suffering from experimental autoimmune encephalomyelitis (EAE), despite receptors maintain their preferential internal distribution. Receptor changes recover following chronic oral therapeutic FTY720 (0.03 mg/Kg/day). These results improve our knowledge of the role of presynaptic release-regulating S1P1Rs and S1P3Rs controlling glutamate transmission in the CNS also unravelling functional adaptations during EAE that recover following chronic FTY720. In a whole, these findings provide new information on the central neuroprotectant activities of FTY720. [ABSTRACT FROM AUTHOR]

Aberrant activation of presynaptic NMDARs in the spinal dorsal horn is integral to opioid-induced hyperalgesia and analgesic tolerance. However, the signaling mechanisms responsible for opioid-induced NMDAR hyperactivity remain poorly identified. Here, we show that repeated treatment with morphine or fentanyl reduced monomeric mGluR5 protein levels in the dorsal root ganglion (DRG) but increased levels of mGluR5 monomers and homodimers in the spinal cord in mice and rats of both sexes. Coimmunoprecipitation analysis revealed that monomeric and dimeric mGluR5 in the spinal cord, but not monomeric mGluR5 in the DRG, directly interacted with GluN1. By contrast, mGluR5 did not interact with l-opioid receptors in the DRG or spinal cord. Repeated morphine treatment markedly increased the mGluR5-GluN1 interaction and protein levels of mGluR5 and GluN1 in spinal synaptosomes. The mGluR5 antagonist MPEP reversed morphine treatment-augmented mGluR5-GluN1 interactions, GluN1 synaptic expression, and dorsal root-evoked monosynaptic EPSCs of dorsal horn neurons. Furthermore, CRISPR-Cas9-induced conditional mGluR5 knockdown in DRG neurons normalized mGluR5 levels in spinal synaptosomes and NMDAR-mediated EPSCs of dorsal horn neurons increased by morphine treatment. Correspondingly, intrathecal injection of MPEP or conditional mGluR5 knockdown in DRG neurons not only potentiated the acute analgesic effect of morphine but also attenuated morphine treatment-induced hyperalgesia and tolerance. Together, our findings suggest that opioid treatment promotes mGluR5 trafficking from primary sensory neurons to the spinal dorsal horn. Through dimerization and direct interaction with NMDARs, presynaptic mGluR5 potentiates and/or stabilizes NMDAR synaptic expression and activity at primary afferent central terminals, thereby maintaining opioid-induced hyperalgesia and tolerance. [ABSTRACT FROM AUTHOR]

Background: The amyloid precursor protein (APP), a key player in Alzheimer's disease (AD), is part of a larger gene family, including the APP like proteins APLP1 and APLP2. They share similar structures, form homo- and heterotypic dimers and exhibit overlapping functions. Results: We investigated complex formation of the APP family members via two inducible dimerization systems, the FKBP-rapamycin based dimerization as well as cysteine induced dimerization, combined with co-immunoprecipitations and Blue Native (BN) gel analyses. Within the APP family, APLP1 shows the highest degree of dimerization and high molecular weight (HMW) complex formation. Interestingly, only about 20% of APP is dimerized in cultured cells whereas up to 50% of APP is dimerized in mouse brains, independent of age and splice forms. Furthermore, we could show that dimerized APP originates mostly from neurons and is enriched in synaptosomes. Finally, BN gel analysis of human cortex samples shows a significant decrease of APP dimers in AD patients compared to controls. Conclusions: Together, we suggest that loss of full-length APP dimers might correlate with loss of synapses in the process of AD. [ABSTRACT FROM AUTHOR]

Synaptic disturbances in excitatory to inhibitory (E/I) balance in forebrain circuits are thought to contribute to the progression of Alzheimer's disease (AD) and dementia, although direct evidence for such imbalance in humans is lacking. We assessed anatomical and electrophysiological synaptic E/I ratios in post-mortem parietal cortex samples from middle-aged individuals with AD (early-onset) or Down syndrome (DS) by fluorescence deconvolution tomography and microtransplantation of synaptic membranes. Both approaches revealed significantly elevated E/I ratios for AD, but not DS, versus controls. Gene expression studies in an independent AD cohort also demonstrated elevated E/I ratios in individuals with AD as compared to controls. These findings provide evidence of a marked pro-excitatory perturbation of synaptic E/I balance in AD parietal cortex, a region within the default mode network that is overly active in the disorder, and support the hypothesis that E/I imbalances disrupt cognition-related shifts in cortical activity which contribute to the intellectual decline in AD.

Synaptosomes are subcellular components isolated from nerve terminations that can be prepared by homogenizing brain tissue in isotonic sucrose solution followed by appropriate centrifugation. Their preparation technique has a long history since synaptosomes were first isolated from nerve endings and described by Gray and Whittaker in 1962. The preparation of synaptosomes produces presynaptic boutons alone or in combination with fragments of postsynaptic membranes. Interestingly, synaptosomes contain organelles and vesicles that express native channels, receptors, and transporters. At 37 °C, these isolated nerve endings are metabolically active and synthesize and release neurotransmitters. They are actively used to investigate neurotransmission, its actors, and the mechanisms of neurotransmitter release. To date, many functional and non-functional applications of synaptosomes have been documented. Due to their versatility, synaptosomes have been actively used to study neuroinflammatory processes.

The pathological hallmark of Parkinson's disease (PD) is the loss of neuromelanin-containing dopaminergic neurons within the substantia nigra pars compacta (SNpc). Additionally, numerous studies indicate an altered synaptic function during disease progression. To gain new insights into the molecular processes underlying the alteration of synaptic function in PD, a proteomic study was performed. Therefore, synaptosomes were isolated by density gradient centrifugation from SNpc tissue of individuals at advanced PD stages (N = 5) as well as control subjects free of pathology (N = 5) followed by mass spectrometry-based analysis. In total, 362 proteins were identified and assigned to the synaptosomal core proteome. This core proteome comprised all proteins expressed within the synapses without regard to data analysis software, gender, age, or disease. The differential analysis between control subjects and PD cases revealed that CD9 antigen was overrepresented and fourteen proteins, among them Thymidine kinase 2 (TK2), mitochondrial, 39S ribosomal protein L37, neurolysin, and Methionine-tRNA ligase (MARS2) were underrepresented in PD suggesting an alteration in mitochondrial translation within synaptosomes.

Glutamate is a major excitatory neurotransmitter that mediates neuronal damage in acute and chronic brain disorders. The effect and mechanism of phillygenin, a natural compound with neuroprotective potential, on glutamate release in isolated nerve terminals (synaptosomes) prepared from the rat cerebral cortex were examined. In this study, 4-aminopyridine (4-AP), a potassium channel blocker, was utilized to induce the release of glutamate, which was subsequently quantified via a fluorometric assay. Our findings revealed that phillygenin reduced 4-AP-induced glutamate release, and this inhibitory effect was reversed by removing extracellular Ca2+ or inhibiting vesicular transport with bafilomycin A1. However, exposure to the glutamate transporter inhibitor dl-threo-beta-benzyl-oxyaspartate (dl-TOBA) did not influence the inhibitory effect. Moreover, phillygenin did not change the synaptosomal membrane potential but lowered the 4-AP-triggered increase in intrasynaptosomal Ca2+ concentration ([Ca2+]i). Antagonizing Cav2.2 (N-type) calcium channels blocked the inhibition of glutamate release by phillygenin, whereas pretreatment with the mitochondrial Na+/Ca2+ exchanger inhibitor, CGP37157 or the ryanodine receptor inhibitor, dantrolene, both of which block intracellular Ca2+ release, had no effect. The effect of phillygenin on glutamate release triggered by 4-AP was completely abolished when MAPK/ERK inhibitors were applied. Furthermore, phillygenin attenuated the phosphorylation of ERK1/2 and its major presynaptic target, synapsin I, a protein associated with synaptic vesicles. These data collectively suggest that phillygenin mediates the inhibition of evoked glutamate release from synaptosomes primarily by reducing the influx of Ca2+ through Cav2.2 calcium channels, thereby subsequently suppressing the MAPK/ERK/synapsin I signaling cascade.

A comprehensive review was conducted to compile the contributions of Mary B. Dratman and studies by other researchers in the field of nongenomic actions of thyroid hormones in adult mammalian brain. Dratman and her collaborators authored roughly half of the papers in this area. It has been almost fifty years since Dratman introduced the novel concept of thyroid hormones as neurotransmitters for the first time. The characterization of unique brain-region specific accumulation of thyroid hormones within the nerve terminals in adult mammals was a remarkable contribution by Dratman. It suggested a neurotransmitter- or neuromodulator-like role of thyroid hormone and/or its derivative, 3-iodothyronamine within adrenergic systems in adult mammalian brain. Several studies by other researchers using synaptosomes as a model system, have contributed to the concept of direct nongenomic actions of thyroid hormones at synaptic regions by establishing that thyroid hormones or their derivatives can bind to synaptosomal membranes, alter membrane functions including enzymatic activities and ion transport, elicit Ca2+/NO-dependent signaling pathways and induce substrate-protein phosphorylation. Such findings can help to explain the physiological and pathophysiological roles of thyroid hormone in psychobehavioral control in adult mammalian brain. However, the exact mode of nongenomic actions of thyroid hormones at nerve terminals in adult mammalian brain awaits further study.

Background: Synaptic changes occur early in patients with Huntington's disease (HD) and in mouse models of HD. An analysis of synaptic changes in HD transgenic sheep (OVT73) is fitting since they have been shown to have some phenotypes. They also have larger brains, longer lifespan, and greater motor and cognitive capacities more aligned with humans, and can provide abundant biofluids for in vivo monitoring of therapeutic interventions. Objective: The objective of this study was to determine if there were differences between 5- and 10-year-old OVT73 and wild-type (WT) sheep in levels of synaptic proteins in brain and in neurofilament light chain (NfL) in cerebrospinal fluid (CSF) and plasma. Methods: Mutant huntingtin (mHTT) and other proteins were measured by western blot assay in synaptosomes prepared from caudate, motor, and piriform cortex in 5-year-old and caudate, putamen, motor; and piriform cortex in 10-year-old WT and OVT73 sheep. Levels of NfL, a biomarker for neuronal damage increased in many neurological disorders including HD, were examined in CSF and plasma samples from 10-year-old WT and OVT73 sheep using the Simoa NfL Advantage kit. Results: Western blot analysis showed mHTT protein expression in synaptosomes from OVT73 sheep was 23% of endogenous sheep HTT levels at both ages. Significant changes were detected in brain levels of PDE10A, SCN4B, DARPP32, calmodulin, SNAP25, PSD95, VGLUT 1, VAMP1, and Na+/K+-ATPase, which depended on age and brain region. There was no difference in NfL levels in CSF and plasma in OVT73 sheep compared to age-matched WT sheep. Conclusions: These results show that synaptic changes occur in brain of 5- and 10-year-old OVT73 sheep, but levels of NfL in biofluids are unaffected. Altogether, the data support a prodromal disease state in OVT73 sheep that involves the caudate, putamen and cortex. [ABSTRACT FROM AUTHOR]

Definition: Synaptosomes are subcellular components isolated from nerve terminations that can be prepared by homogenizing brain tissue in isotonic sucrose solution followed by appropriate centrifugation. Their preparation technique has a long history since synaptosomes were first isolated from nerve endings and described by Gray and Whittaker in 1962. The preparation of synaptosomes produces presynaptic boutons alone or in combination with fragments of postsynaptic membranes. Interestingly, synaptosomes contain organelles and vesicles that express native channels, receptors, and transporters. At 37 °C, these isolated nerve endings are metabolically active and synthesize and release neurotransmitters. They are actively used to investigate neurotransmission, its actors, and the mechanisms of neurotransmitter release. To date, many functional and non-functional applications of synaptosomes have been documented. Due to their versatility, synaptosomes have been actively used to study neuroinflammatory processes. [ABSTRACT FROM AUTHOR]

The involvement of mitochondrial dysfunction in cystatin B (CSTB) deficiency has been suggested, but its role in the onset of neurodegeneration, myoclonus, and ataxia in the CSTB-deficient mouse model (Cstb-/-) is yet unknown. CSTB is an inhibitor of lysosomal and nuclear cysteine cathepsins. In humans, partial loss-offunction mutations cause the progressive myoclonus epilepsy neurodegenerative disorder, EPM1. Here we applied proteome analysis and respirometry on cerebellar synaptosomes from early symptomatic (Cstb-/-) mice to identify the molecular mechanisms involved in the onset of CSTB-deficiency associated neural pathogenesis. Proteome analysis showed that CSTB deficiency is associated with differential expression of mitochondrial and synaptic proteins, and respirometry revealed a progressive impairment in mitochondrial function coinciding with the onset of myoclonus and neurodegeneration in (Cstb-/-) mice. This mitochondrial dysfunction was not associated with alterations in mitochondrial DNA copy number or membrane ultrastructure. Collectively, our results show that CSTB deficiency generates a defect in synaptic mitochondrial bioenergetics that coincides with the onset and progression of the clinical phenotypes, and thus is likely a contributor to the pathogenesis of EPM1. [ABSTRACT FROM AUTHOR]

Individual variation in the addiction liability of amphetamines has a heritable genetic component. We previously identified Hnrnph1 (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene underlying decreased methamphetamine-induced locomotor activity in mice. Here, we showed that mice (both females and males) with a heterozygous mutation in the first coding exon of Hnrnph1 (H1+/-) showed reduced methamphetamine reinforcement and intake and dose-dependent changes in methamphetamine reward as measured via conditioned place preference. Furthermore, H1+/- mice showed a robust decrease in methamphetamine-induced dopamine release in the NAc with no change in baseline extracellular dopamine, striatal whole-tissue dopamine, dopamine transporter protein, dopamine uptake, or striatal methamphetamine and amphetamine metabolite levels. Immunohistochemical and immunoblot staining of midbrain dopaminergic neurons and their forebrain projections for TH did not reveal any major changes in staining intensity, cell number, or forebrain puncta counts. Surprisingly, there was a twofold increase in hnRNP H protein in the striatal synaptosome of H1+/- mice with no change in whole-tissue levels. To gain insight into the mechanisms linking increased synaptic hnRNP H with decreased methamphetamine-induced dopamine release and behaviors, synaptosomal proteomic analysis identified an increased baseline abundance of several mitochondrial complex I and V proteins that rapidly decreased at 30 min after methamphetamine administration in H1+/- mice. In contrast, the much lower level of basal synaptosomal mitochondrial proteins in WT mice showed a rapid increase. We conclude that H1+/- decreases methamphetamine-induced dopamine release, reward, and reinforcement and induces dynamic changes in basal and methamphetamine-induced synaptic mitochondrial function.SIGNIFICANCE STATEMENT Methamphetamine dependence is a significant public health concern with no FDA-approved treatment. We discovered a role for the RNA binding protein hnRNP H in methamphetamine reward and reinforcement. Hnrnph1 mutation also blunted methamphetamine-induced dopamine release in the NAc, a key neurochemical event contributing to methamphetamine addiction liability. Finally, Hnrnph1 mutants showed a marked increase in basal level of synaptosomal hnRNP H and mitochondrial proteins that decreased in response to methamphetamine, whereas WT mice showed a methamphetamine-induced increase in synaptosomal mitochondrial proteins. Thus, we identified a potential role for hnRNP H in basal and dynamic mitochondrial function that informs methamphetamine-induced cellular adaptations associated with reduced addiction liability.

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. Active m-calpain is present in the media generated from striatal synaptosomes. Glyburide prevents m-calpain release from striatal synaptosomes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Apolipoprotein E (apoE) colocalizes with amyloid-β (Aβ) in Alzheimer disease (AD) plaques and in synapses, and evidence suggests that direct interactions between apoE and Aβ are important for apoE's effects in AD. The present work examines the hypothesis that apoE receptors mediate uptake of apoE/Aβ complex into synaptic terminals. Western blot analysis shows multiple SDS-stable assemblies in synaptosomes from human AD cortex; apoE/Aβ complex was markedly increased in AD compared with aged control samples. Complex formation between apoE and Aβ was confirmed by coimmunoprecipitation experiments. The apoE receptors low-density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1) were quantified in synaptosomes using flow cytometry, revealing up-regulation of LRP1 in early- and late-stage AD. Dual-labeling flow cytometry analysis of LRP1- and LDLR positives indicate most (approximately 65%) of LDLR and LRP1 is associated with postsynaptic density-95 (PSD-95)-positive synaptosomes, indicating that remaining LRP1 and LDLR receptors are exclusively presynaptic. Flow cytometry analysis of Nile red labeling revealed a reduction in cholesterol esters in AD synaptosomes. Dual-labeling experiments showed apoE and Aβ concentration into LDLR and LRP1-positive synaptosomes, along with free and esterified cholesterol. Synaptic Aβ was increased by apoE4 in control and AD samples. These results are consistent with uptake of apoE/Aβ complex and associated lipids into synaptic terminals, with subsequent Aβ clearance in control synapses and accumulation in AD synapses.

RationaleOver the last decade, many new psychostimulant analogues have appeared on the recreational drug market and most are derivatives of amphetamine or cathinone. Another class of designer drugs is derived from the 2-aminoindan structural template. Several members of this class, including the parent compound 2-aminoindan (2-AI), have been sold as designer drugs. Another aminoindan derivative, 5-methoxy-2-aminoindan (5-MeO-AI or MEAI), is the active ingredient in a product marketed online as an alcohol substitute.MethodsHere, we tested 2-AI and its ring-substituted derivatives 5-MeO-AI, 5-methoxy-6-methyl-2-aminoindan (MMAI), and 5,6-methylenedioxy-2-aminoindan (MDAI) for their abilities to interact with plasma membrane monoamine transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). We also compared the binding affinities of the aminoindans at 29 receptor and transporter binding sites.Results2-AI was a selective substrate for NET and DAT. Ring substitution increased potency at SERT while reducing potency at DAT and NET. MDAI was moderately selective for SERT and NET, with tenfold weaker effects on DAT. 5-MeO-AI exhibited some selectivity for SERT, having sixfold lower potency at NET and 20-fold lower potency at DAT. MMAI was highly selective for SERT, with 100-fold lower potency at NET and DAT. The aminoindans had relatively high affinity for α2-adrenoceptor subtypes. 2-AI had particularly high affinity for α2C receptors (Ki = 41 nM) and slightly lower affinity for the α2A (Ki = 134 nM) and α2B (Ki = 211 nM) subtypes. 5-MeO-AI and MMAI also had moderate affinity for the 5-HT2B receptor.Conclusions2-AI is predicted to have (+)-amphetamine-like effects and abuse potential whereas the ring-substituted derivatives may produce 3,4-methylenedioxymethamphetamine (MDMA)-like effects but with less abuse liability.

CNS inflammatory responses are linked to cognitive impairment in humans. Research in animal models supports this connection by showing that inflammatory cytokines suppress long-term potentiation (LTP), the best-known cellular correlate of memory. Cytokine-induced modulation of LTP has been previously studied in vivo or in brain slices, two experimental approaches containing multiple cell populations responsive to cytokines. In their target cells, cytokines commonly increase the expression of multiple cytokines, thus increasing the complexity of brain cytokine networks even after single-cytokine challenges. Whether cytokines suppress LTP by direct effects on neurons or by indirect mechanisms is still an open question. Here, we evaluated the effect of a major set of inflammatory cytokines including tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β) and interleukin-18 (IL-18) on chemically-induced LTP (cLTP) in isolated hippocampal synaptosomes of mice, using fluorescence analysis of single-synapse long-term potentiation (FASS-LTP). We found that TNFα and IL-1β suppress synaptosomal cLTP. In contrast, cLTP was not affected by IL-18, at a concentration previously shown to block LTP in hippocampal slices. We also found that IL-18 does not impair cLTP or brain-derived neurotrophic factor (BDNF) signaling in primary hippocampal neuronal cultures. Thus, using both synaptosomes and neuron cultures, our data suggest that IL-18 impairs LTP by indirect mechanisms, which may depend on non-neuronal cells, such as glia. Notably, our results demonstrate that TNFα and IL-1β directly suppress hippocampal plasticity via neuron-specific mechanisms. A better understanding of the brain's cytokine networks and their final molecular effectors is crucial to identify specific targets for intervention.

Synapses are integral for healthy brain function and are becoming increasingly recognized as key structures in the early stages of brain disease. Understanding the pathological processes driving synaptic dysfunction will unlock new therapeutic opportunities for some of the most devastating diseases of our time. To achieve this we need a solid repertoire of imaging and molecular tools to interrogate synaptic biology at greater resolution. Synapses have historically been examined in small numbers, using highly technical imaging modalities, or in bulk, using crude molecular approaches. However, recent advances in imaging techniques are allowing us to analyze large numbers of synapses, at single-synapse resolution. Furthermore, multiplexing is now achievable with some of these approaches, meaning we can examine multiple proteins at individual synapses in intact tissue. New molecular techniques now allow accurate quantification of proteins from isolated synapses. The development of increasingly sensitive mass-spectrometry equipment means we can now scan the synaptic molecular landscape almost in totality and see how this changes in disease. As we embrace these new technical developments, synapses will be viewed with clearer focus, and the field of synaptopathy will become richer with insightful and high-quality data. Here, we will discuss some of the ways in which synaptic interrogation is being facilitated by methodological advances, focusing on imaging, and mass spectrometry.

Oxidative stress is one of the key factors responsible for many diseases–neurodegenerative (Parkinson and Alzheimer) diseases, diabetes, atherosclerosis, etc. Creatine, a natural amino acid derivative, is capable of exerting mild, direct antioxidant activity in cultured mammalian cells acutely injured with an array of different reactive oxygen species (ROS) generating compounds. The aim of the study was in vitro (on isolated rat brain sub-cellular fractions–synaptosomes, mitochondria and microsomes) evaluation of newly synthetized creatine derivatives for possible antioxidant and neuroprotective activity. The synaptosomes and mitochondria were obtained by multiple centrifugations with Percoll, while microsomes–only by multiple centrifugations. Varying models of oxidative stress were used to study the possible antioxidant and neuroprotective effects of the respective compounds: on synaptosomes–6-hydroxydopamine; on mitochondria–tert-butyl hydroperoxide; and on microsomes–iron/ascorbate (non-enzyme-induced lipid peroxidation). Administered alone, creatine derivatives and creatine (at concentration 38 µM) revealed neurotoxic and pro-oxidant effects on isolated rat brain subcellular fractions (synaptosomes, mitochondria and microsomes). In models of 6-hydroxydopamine (on synaptosomes), tert-butyl hydroperoxide (on mitochondria) and iron/ascorbate (on microsomes)-induced oxidative stress, the derivatives showed neuroprotective and antioxidant effects. These effects may be due to the preservation of the reduced glutathione level, ROS scavenging and membranes' stabilizers against free radicals. Thus, they play a role in the antioxidative defense system and have a promising potential as therapeutic neuroprotective agents for the treatment of neurodegenerative disorders, connected with oxidative stress, such as Parkinson's disease. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • ALS2 forms a unique homophilic high-molecular weight complex in synaptosomes. • ALS2 exists as a tetramer in cytosol in the brain and liver. • Formation of the ALS2 complex is independent of neural development stages. [ABSTRACT FROM AUTHOR]

It is widely accepted that some types of learning involve structural and functional changes of hippocampal synapses. Cell adhesion molecules neural cell adhesion molecule (NCAM), its polysialylated form polysialic acid to NCAM (PSA-NCAM), and L1 are prominent modulators of those changes. On the other hand, trace eyeblink conditioning, an associative motor learning task, requires the active participation of hippocampal circuits. However, the involvement of NCAM, PSA-NCAM, and L1 in this type of learning is not fully known. Here, we aimed to investigate the possible time sequence modifications of such neural cell adhesion molecules in the hippocampus during the acquisition of a trace eyeblink conditioning. To do so, the hippocampal expression of NCAM, PSA-NCAM, and L1 was assessed at three different time points during conditioning: after one (initial acquisition), three (partial acquisition), and six (complete acquisition) sessions of the conditioning paradigm. The conditioned stimulus (CS) was a weak electrical pulse separated by a 250-ms time interval from the unconditioned stimuli (US, a strong electrical pulse). An acquisition-dependent regulation of these adhesion molecules was found in the hippocampus. During the initial acquisition of the conditioning eyeblink paradigm (12 h after 1 and 3 days of training), synaptic expression of L1 and PSA-NCAM was transiently increased in the contralateral hippocampus to the paired CS-US presentations, whereas, when the associative learning was completed, such increase disappeared, but a marked and bilateral upregulation of NCAM was found. In conclusion, our findings show a specific temporal pattern of hippocampal CAMs expression during the acquisition process, highlighting the relevance of NCAM, PSA-NCAM, and L1 as learning-modulated molecules critically involved in remodeling processes underlying associative motor-memories formation. [ABSTRACT FROM AUTHOR]

The racemic mixture dimiracetam negatively modulates NMDA-induced glutamate release in rat spinal cord synaptosomal preparations and is orally effective in models of neuropathic pain. In this study, we compared the effects of dimiracetam, its R- or S-enantiomers, and the R:S 3:1 non-racemic mixture (MP-101). In vitro, dimiracetam was more potent than its R- or S-enantiomers in reducing the NMDA-induced [3H]D-aspartate release in rat spinal cord synaptosomes. Similarly, acute oral administration of dimiracetam was more effective than a single enantiomer in the sodium monoiodoacetate (MIA) paradigm of painful osteoarthritis. Then, we compared the in vitro effects of a broad range of non-racemic enantiomeric mixtures on the NMDA-induced [3H]D-aspartate release. Dimiracetam was a more potent blocker than each isolated enantiomer but the R:S 3:1 non-racemic mixture (MP-101) was even more potent than dimiracetam, with an IC50 in the picomolar range. In the chronic oxaliplatin-induced neuropathic pain model, MP-101 showed a significantly improved anti-neuropathic profile, and its effect continued one week after treatment suspension. MP-101 also performed better than dimiracetam in animal models of cognition and depression. Based on the benign safety and tolerability profile previously observed with racemic dimiracetam, MP-101 appears to be a novel, promising clinical candidate for the prevention and treatment of several neuropathic and neurological disorders. [ABSTRACT FROM AUTHOR]

Rat brain synaptosomes were studied with ART-IR spectroscopy and confocal microscopy. The isolated synaptosomes visualized with confocal microscopy look like integral subcellular structures of 0.5-2 μm in size, which correlates with the literature data. The spectroscopic signatures of synaptosomes incubated at different temperatures: −20, 22, 37, and 42 °C, have been identified in the 650-3700 cm−1 region. Significant changes in the hydrogen bonded N-H, PO2-, and C-N-C groups with temperature are demonstrated. [ABSTRACT FROM AUTHOR]

3,4-Methylenedioximethamphetamine (MDMA; "ecstasy") is a psychotropic drug with well-known neurotoxic effects mediated by hitherto not fully understood mechanisms. The Na+- and K+-activated adenosine 5′-triphosphatase (Na+/K+ ATPase), by maintaining the ion gradient across the cell membrane, regulates neuronal excitability. Thus, a perturbation of its function strongly impacts cell homeostasis, ultimately leading to neuronal dysfunction and death. Nevertheless, whether MDMA affects the Na+/K+ ATPase remains unknown. In this study, we used synaptosomes obtained from whole mouse brain to test the effects of MDMA, three of its major metabolites [α-methyldopamine, N-methyl-α-methyldopamine and 5-(glutathion-S-yl)-α-methyldopamine], serotonin (5-HT), dopamine, 3,4-dihydroxy-l-phenylalanine (l-Dopa) and 3,4-dihydroxyphenylacetic acid (DOPAC) on the Na+/K+ ATPase function. A concentration-dependent increase of Na+/K+ ATPase activity was observed in synaptosomes exposed to the tested compounds (concentrations ranging from 0.0625 to 200 µM). These effects were independent of protein kinases A and C activities. Nevertheless, a rescue of the compounds' effects was observed in synaptosomes pre-incubated with the antioxidant N-acetylcysteine (1 mM), suggesting a role for reactive species-regulated pathways on the Na+/K+ ATPase effects. In agreement with this hypothesis, a similar increase in the pump activity was found in synaptosomes exposed to the chemical generator of superoxide radicals, phenazine methosulfate (1–250 µM). This study demonstrates the ability of MDMA metabolites, monoamine neurotransmitters, l-Dopa and DOPAC to alter the Na+/K+ ATPase function. This could represent a yet unknown mechanism of action of MDMA and its metabolites in the brain. [ABSTRACT FROM AUTHOR]

Chronic social isolation (CSIS)–induced alternation in synaptic and mitochondrial function of specific brain regions is associated with major depressive disorder (MDD). Despite the wide number of available medications, treating MDD remains an important challenge. Although fluoxetine (Flx) is the most frequently prescribed antidepressant, its mode of action is still unknown. To delineate affected molecular pathways of depressive-like behavior and identify potential targets upon Flx treatment, we performed a comparative proteomic analysis of hippocampal purified synaptic terminals (synaptosomes) of rats exposed to six weeks of CSIS, an animal model of depression, and/or followed by Flx treatment (lasting three weeks of six-week CSIS) to explore synaptic protein profile changes. Results showed that Flx in controls mainly induced decreased expression of proteins involved in energy metabolism and the redox system. CSIS led to increased expression of proteins that mainly participate in Ca2+/calmodulin-dependent protein kinase II (Camk2)-related neurotransmission, vesicle transport, and ubiquitination. Flx treatment of CSIS rats predominantly increased expression of proteins involved in synaptic vesicle trafficking (exocytosis and endocytosis), and energy metabolism (glycolytic and mitochondrial respiration). Overall, these Flx-regulated changes in synaptic and mitochondrial proteins of CSIS rats might be critical targets for new therapeutic development for the treatment of MDD. [ABSTRACT FROM AUTHOR]

Abstract—We collected data on the development of Rudolf Nikolaevich Glebov's scientific school in neurochemistry. We reviewed the main achievements of this school in the field of biochemistry of synapses, studies on the activity of synaptic ATPases, parameters of the functioning of ionic channels, the mechanisms of coupling of membrane depolarization and secretion of transmitters, and the pathogenesis of epilepsy. We presented the main works by R.N. Glebov as well as the memories of his colleagues about this amazing scientist and broad-minded person. [ABSTRACT FROM AUTHOR]