Akt activation ameliorates deficits in hippocampal-dependent memory and activity-dependent synaptic protein synthesis in an Alzheimer's disease mouse model.

Protein kinase-B (Akt) and the mechanistic target of rapamycin (mTOR) signaling pathways are implicated in Alzheimer's disease (AD) pathology. Akt/mTOR signaling pathways, activated by external inputs, enable new protein synthesis at the synapse and synaptic plasticity. The molecular mechanisms impeding new protein synthesis at the synapse in AD pathogenesis remain elusive. Here, we aimed to understand the molecular mechanisms prior to the manifestation of histopathological hallmarks by characterizing Akt1/mTOR signaling cascades and new protein synthesis in the hippocampus of WT and amyloid precursor protein/presenilin-1 (APP/PS1) male mice. Intriguingly, compared to those in WT mice, we found significant decreases in pAkt1, pGSK3ß, pmTOR, pS6 ribosomal protein, and p4E-BP1 levels in both post nuclear supernatant and synaptosomes isolated from the hippocampus of one-month-old (presymptomatic) APP/PS1 mice. In synaptoneurosomes prepared from the hippocampus of presymptomatic APP/PS1 mice, activity-dependent protein synthesis at the synapse was impaired and this deficit was sustained in young adults. In hippocampal neurons from C57BL/6 mice, downregulation of Akt1 precluded synaptic activity-dependent protein synthesis at the dendrites but not in the soma. In three-month-old APP/PS1 mice, Akt activator (SC79) administration restored deficits in memory recall and activity-dependent synaptic protein synthesis. C57BL/6 mice administered with an Akt inhibitor (MK2206) resulted in memory recall deficits compared to those treated with vehicle. We conclude that dysregulation of Akt1/mTOR and its downstream signaling molecules in the hippocampus contribute to memory recall deficits and loss of activity-dependent synaptic protein synthesis. In AD mice, however, Akt activation ameliorates deficits in memory recall and activity-dependent synaptic protein synthesis.

Cystatin C loaded in brain-derived extracellular vesicles rescues synapses after ischemic insult in vitro and in vivo.

Synaptic loss is an early event in the penumbra area after an ischemic stroke. Promoting synaptic preservation in this area would likely improve functional neurological recovery. We aimed to detect proteins involved in endogenous protection mechanisms of synapses in the penumbra after stroke and to analyse potential beneficial effects of these candidates for a prospective stroke treatment. For this, we performed Liquid Chromatography coupled to Mass Spectrometry (LC-MS)-based proteomics of synaptosomes isolated from the ipsilateral hemispheres of mice subjected to experimental stroke at different time points (24 h, 4 and 7 days) and compared them to sham-operated mice. Proteomic analyses indicated that, among the differentially expressed proteins between the two groups, cystatin C (CysC) was significantly increased at 24 h and 4 days following stroke, before returning to steady-state levels at 7 days, thus indicating a potential transient and intrinsic rescue mechanism attempt of neurons. When CysC was applied to primary neuronal cultures subjected to an in vitro model of ischemic damage, this treatment significantly improved the preservation of synaptic structures. Notably, similar effects were observed when CysC was loaded into brain-derived extracellular vesicles (BDEVs). Finally, when CysC contained in BDEVs was administered intracerebroventricularly to stroked mice, it significantly increased the expression of synaptic markers such as SNAP25, Homer-1, and NCAM in the penumbra area compared to the group supplied with empty BDEVs. Thus, we show that CysC-loaded BDEVs promote synaptic protection after ischemic damage in vitro and in vivo, opening the possibility of a therapeutic use in stroke patients.

Density of Sphingosine-1-Phosphate Receptors Is Altered in Cortical Nerve-Terminals of Insulin-Resistant Goto-Kakizaki Rats and Diet-Induced Obese Mice.

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.

STIM2 regulates NMDA receptor endocytosis that is induced by short-term NMDA receptor overactivation in cortical neurons.

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.

Unique Properties of Synaptosomes and Prospects for Their Use for the Treatment of Alzheimer's Disease.

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.

Early-life stress and amyloidosis in mice share pathogenic pathways involving synaptic mitochondria and lipid metabolism.

INTRODUCTION: Early-life stress (ES) increases the risk for Alzheimer's disease (AD). We and others have shown that ES aggravates amyloid-beta (Aß) 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.

Neuroanatomical zones of human traumatic brain injury reveal significant differences in protein profile and protein oxidation: Implications for secondary injury events.

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.

Increased hippocampal cannabinoid 1 receptor expression is associated with protection from severe seizures in pregnant mice with reduced uterine perfusion pressure.

Eclampsia, new-onset seizures in pregnancy, can complicate preeclampsia, a hypertensive pregnancy disorder. The mechanisms contributing to increased risk of seizures in preeclampsia are not fully known. One mechanism could be abnormal endocannabinoid system (ECS) activity and impaired neuromodulation. Indeed, increased placental cannabinoid receptor 1 (CB1R) expression and reduced serum anandamide, a CB1R ligand, have been reported in preeclampsia patients. We hypothesized that reduced uterine perfusion pressure (RUPP), used to mimic preeclampsia, leads to changes in hippocampal CB1R expression, and that manipulating CB1R activity will change seizure severity in RUPP mice. Pregnant mice underwent sham or RUPP surgery on gestational day (GD)13.5. On GD18.5, mice received: no drug treatment, pentylenetetrazol (PTZ, 40 mg/kg), Rimonabant (10 mg/kg) + PTZ, or 2-AG (1 mg/kg) + PTZ. Behaviors were video recorded (15 min for Rimonabant and 2-AG, followed by 30 min for PTZ), and the hippocampus was harvested. The expression of CB1R and ECS proteins was measured in hippocampal homogenates, synaptosomes, and cytosol. Hippocampal CB1R increased in homogenates and cytosolic fraction, and was unchanged in synaptosomes of RUPP mice. Increased CB1R colocalization on glutamate-releasing neurons within hippocampal CA1 was observed in RUPP mice. Rimonabant modestly increased seizure scores over time in RUPP mice. PTZ after rimonabant pretreatment increased seizure scores and duration, while reducing latency in sham mice, with little to no change in RUPP mice. Furthermore, RUPP mice had lower seizure scores over time than sham following CB1R blockade and activation. These data suggest that RUPP modifies CB1R activity prior to seizure induction, which protects mice from worse seizure outcomes.

Feedback facilitation by adenosine A2A receptors of ATP release from mouse hippocampal nerve terminals.

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,ß-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.

Aß/tau oligomer interplay at human synapses supports shifting therapeutic targets for Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is characterized by progressive cognitive decline due to accumulating synaptic insults by toxic oligomers of amyloid beta (AßO) and tau (TauO). There is growing consensus that preventing these oligomers from interacting with synapses might be an effective approach to treat AD. However, recent clinical trial failures suggest low effectiveness of targeting Aß in late-stage AD. Researchers have redirected their attention toward TauO as the levels of this species increase later in disease pathogenesis. Here we show that AßO and TauO differentially target synapses and affect each other's binding dynamics. METHODS: Binding of labeled, pre-formed Aß and tau oligomers onto synaptosomes isolated from the hippocampus and frontal cortex of mouse and postmortem cognitively intact elderly human brains was evaluated using flow-cytometry and western blot analyses. Binding of labeled, pre-formed Aß and tau oligomers onto mouse primary neurons was assessed using immunofluorescence assay. The synaptic dysfunction was measured by fluorescence analysis of single-synapse long-term potentiation (FASS-LTP) assay. RESULTS: We demonstrated that higher TauO concentrations effectively outcompete AßO and become the prevailing synaptic-associated species. Conversely, high concentrations of AßO facilitate synaptic TauO recruitment. Immunofluorescence analyses of mouse primary cortical neurons confirmed differential synaptic binding dynamics of AßO and TauO. Moreover, in vivo experiments using old 3xTgAD mice ICV injected with either AßO or TauO fully supported these findings. Consistent with these observations, FASS-LTP analyses demonstrated that TauO-induced suppression of chemical LTP was exacerbated by AßO. Finally, predigestion with proteinase K abolished the ability of TauO to compete off AßO without affecting the ability of high AßO levels to increase synaptic TauO recruitment. Thus, unlike AßO, TauO effects on synaptosomes are hampered by the absence of protein substrate in the membrane. CONCLUSIONS: These results introduce the concept that TauO become the main synaptotoxic species at late AD, thus supporting the hypothesis that TauO may be the most effective therapeutic target for clinically manifest AD.

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.

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.

Neuroprotective evaluation of novel substituted 1,3,4-oxadiazole and aroylhydrazone derivatives.

The paper reports on the facile and convenient synthesis of a series of novel 2,5-substituted 1,3,4-oxadiazoles 3a-f and that of aroylhydrazone-based molecular hybrids 5a-g from readily available starting materials. The structure of the compounds was confirmed by IR, 1H NMR, 13C NMR and HRESI-MS spectral data. The toxicological potential of the compounds was evaluated by monitoring the synaptosomal viability and the levels of reduced glutathione in rat brain synaptosomes, isolated by Percoll gradient. The neuroprotective effects were assessed in vitro in a model of 6-hydroxydopamine-induced neurotoxicity. Administered alone, at a concentration of 40 µM, most of the 1,3,4-oxadiazole derivatives and all of the hydrazone derivatives exhibited weak statistically significant neurotoxic effects, compared to the control. Two of the compounds from the novel oxadiazoles 3a and 3d did not have any toxicity. In a model of 6-OHDA-induced oxidative stress, again 3a and 3d and all aroylhydrazone derivatives 5a-g revealed statistically significant neuroprotective effect by preserving the synaptosomal viability and the level of reduced glutathione, against the toxic agent. Some of the compounds may have neuroprotective effects due to possible stabilization of the synaptosomal membrane and/or because of the preserved reduced glutathione. Additionally, all the compounds display a good predicted ADME profile.

Ecstasy metabolites and monoamine neurotransmitters upshift the Na+/K+ ATPase activity in mouse brain synaptosomes.

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.

Decoding the Synaptic Proteome with Long-Term Exposure to Midazolam during Early Development.

The intensive use of anesthetic and sedative agents in the neonatal intensive care unit (NICU) has raised controversial concerns about the potential neurodevelopmental risks. This study focused on midazolam (MDZ), a common benzodiazepine regularly used as a sedative on neonates in the NICU. Mounting evidence suggests a single exposure to MDZ during the neonatal period leads to learning disturbances. However, a knowledge gap that remains is how long-term exposure to MDZ during very early stages of life impacts synaptic alterations. Using a preclinical rodent model system, we mimicked a dose-escalation regimen on postnatal day 3 (P3) pups until day 21. Next, purified synaptosomes from P21 control and MDZ animals were subjected to quantitative mass-spectrometry-based proteomics, to identify potential proteomic signatures. Further analysis by ClueGO identified enrichment of proteins associated with actin-binding and protein depolymerization process. One potential hit identified was alpha adducin (ADD1), belonging to the family of cytoskeleton proteins, which was upregulated in the MDZ group and whose expression was further validated by Western blot. In summary, this study sheds new information on the long-term exposure of MDZ during the early stages of development impacts synaptic function, which could subsequently perturb neurobehavioral outcomes at later stages of life.

Fluoxetine Enhances Synaptic Vesicle Trafficking and Energy Metabolism in the Hippocampus of Socially Isolated Rats.

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.

An Anthranilate Derivative Inhibits Glutamate Release and Glutamate Excitotoxicity in Rats.

The neurotransmitter glutamate plays an essential role in excitatory neurotransmission; however, excessive amounts of glutamate lead to excitotoxicity, which is the most common pathogenic feature of numerous brain disorders. This study aimed to investigate the role of butyl 2-[2-(2-fluorophenyl)acetamido]benzoate (HFP034), a synthesized anthranilate derivative, in the central glutamatergic system. We used rat cerebro-cortical synaptosomes to examine the effect of HFP034 on glutamate release. In addition, we used a rat model of kainic acid (KA)-induced glutamate excitotoxicity to evaluate the neuroprotective potential of HFP034. We showed that HFP034 inhibits 4-aminopyridine (4-AP)-induced glutamate release from synaptosomes, and this inhibition was absent in the absence of extracellular calcium. HFP034-mediated inhibition of glutamate release was associated with decreased 4-AP-evoked Ca2+ level elevation and had no effect on synaptosomal membrane potential. The inhibitory effect of HFP034 on evoked glutamate release was suppressed by blocking P/Q-type Ca2+ channels and protein kinase C (PKC). Furthermore, HFP034 inhibited the phosphorylation of PKC and its substrate, myristoylated alanine-rich C kinase substrate (MARCKS) in synaptosomes. We also observed that HFP034 pretreatment reduced neuronal death, glutamate concentration, glial activation, and the levels of endoplasmic reticulum stress-related proteins, calpains, glucose-regulated protein 78 (GRP 78), C/EBP homologous protein (CHOP), and caspase-12 in the hippocampus of KA-injected rats. We conclude that HFP034 is a neuroprotective agent that prevents glutamate excitotoxicity, and we suggest that this effect involves inhibition of presynaptic glutamate release through the suppression of P/Q-type Ca2+ channels and PKC/MARCKS pathways.

Differential Ganglioside and Cholesterol Depletion by Various Cyclodextrin Derivatives and Their Effect on Synaptosomal Glutamate Release.

Gangliosides are glycosphingolipids of the plasma membrane and are highly enriched in the nervous system where they play a vital role in normal cell functions. Furthermore, several studies suggest their potential involvement in the pathogenesis of neurological conditions. Since cyclodextrins (CDs) can form inclusion complexes with various lipids, methylated beta-CDs are widely used in biomedical research to extract cholesterol from the membrane and study its cellular role. Despite CDs being known to interact with other membrane lipid components, their effect on gangliosides is poorly characterized. The aim of this research was to investigate the effect of dimethyl-beta-cyclodextrin (DIMEB), hydroxypropyl-beta-cyclodextrin (HPBCD), randomly methylated-alpha-cyclodextrin (RAMEA), and hydroxypropyl-alpha-cyclodextrin (HPACD) on ganglioside and cholesterol levels in rat brain synaptosomes. Their effect on membrane integrity and viability was also assessed. We examined the role of lipid depletion by CDs on the release of the major excitatory neurotransmitter, glutamate. Selective concentration range for cholesterol depletion was only found with HPBCD, but not with DIMEB. Selective depletion of gangliosides was achieved by both RAMEA and HPACD. The inhibition of stimulated glutamate release upon ganglioside depletion was found, suggesting their potential role in neurotransmission. Our study highlights the importance of the characterization of the lipid depleting capability of different CDs.

Inhibition of Glutamate Release from Rat Cortical Nerve Terminals by Dehydrocorydaline, an Alkaloid from Corydalis yanhusuo.

Excessive release of glutamate induces excitotoxicity and causes neuronal damage in several neurodegenerative diseases. Natural products have emerged as potential neuroprotective agents for preventing and treating neurological disorders. Dehydrocorydaline (DHC), an active alkaloid compound isolated from Corydalis yanhusuo, possesses neuroprotective capacity. The present study investigated the effect of DHC on glutamate release using a rat brain cortical synaptosome model. Our results indicate that DHC inhibited 4-aminopyridine (4-AP)-evoked glutamate release and elevated intrasynaptosomal calcium levels. The inhibitory effect of DHC on 4-AP-evoked glutamate release was prevented in the presence of the vesicular transporter inhibitor bafilomycin A1 and the N- and P/Q-type Ca2+ channel blocker ω-conotoxin MVIIC but not the intracellular inhibitor of Ca2+ release dantrolene or the mitochondrial Na+/Ca2+ exchanger inhibitor CGP37157. Moreover, the inhibitory effect of DHC on evoked glutamate release was prevented by the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) inhibitor PD98059. Western blotting data in synaptosomes also showed that DHC significantly decreased the level of ERK1/2 phosphorylation and synaptic vesicle-associated protein synapsin I, the main presynaptic target of ERK. Together, these results suggest that DHC inhibits presynaptic glutamate release from cerebrocortical synaptosomes by suppressing presynaptic voltage-dependent Ca2+ entry and the MAPK/ERK/synapsin I signaling pathway.

Cytosolic, but not matrix, calcium is essential for adjustment of mitochondrial pyruvate supply.

Mitochondrial oxidative phosphorylation (OXPHOS) and cellular workload are tightly balanced by the key cellular regulator, calcium (Ca2+). Current models assume that cytosolic Ca2+ regulates workload and that mitochondrial Ca2+ uptake precedes activation of matrix dehydrogenases, thereby matching OXPHOS substrate supply to ATP demand. Surprisingly, knockout (KO) of the mitochondrial Ca2+ uniporter (MCU) in mice results in only minimal phenotypic changes and does not alter OXPHOS. This implies that adaptive activation of mitochondrial dehydrogenases by intramitochondrial Ca2+ cannot be the exclusive mechanism for OXPHOS control. We hypothesized that cytosolic Ca2+, but not mitochondrial matrix Ca2+, may adapt OXPHOS to workload by adjusting the rate of pyruvate supply from the cytosol to the mitochondria. Here, we studied the role of malate-aspartate shuttle (MAS)-dependent substrate supply in OXPHOS responses to changing Ca2+ concentrations in isolated brain and heart mitochondria, synaptosomes, fibroblasts, and thymocytes from WT and MCU KO mice and the isolated working rat heart. Our results indicate that extramitochondrial Ca2+ controls up to 85% of maximal pyruvate-driven OXPHOS rates, mediated by the activity of the complete MAS, and that intramitochondrial Ca2+ accounts for the remaining 15%. Of note, the complete MAS, as applied here, included besides its classical NADH oxidation reaction the generation of cytosolic pyruvate. Part of this largely neglected mechanism has previously been described as the "mitochondrial gas pedal." Its implementation into OXPHOS control models integrates seemingly contradictory results and warrants a critical reappraisal of metabolic control mechanisms in health and disease.