Human Brain Synaptosomes Research Articles

The expression system influences stability, maturation efficiency, and oligomeric properties of the potassium-chloride co-transporter KCC2

The neuron-specific K+/Cl− co-transporter 2, KCC2, which is critical for brain development, regulates γ-aminobutyric acid-dependent inhibitory neurotransmission. Consistent with its function, mutations in KCC2 are linked to neurodevelopmental disorders, including epilepsy, schizophrenia, and autism. KCC2 possesses 12 transmembrane spans and forms an intertwined dimer. Based on its complex architecture and function, reduced cell surface expression and/or activity have been reported when select disease-associated mutations are present in the gene encoding the protein, SLC12A5. These data suggest that KCC2 might be inherently unstable, as seen for other complex polytopic ion channels, thus making it susceptible to cellular quality control pathways that degrade misfolded proteins. To test these hypotheses, we examined KCC2 stability and/or maturation in five model systems: yeast, HEK293 cells, primary rat neurons, and rat and human brain synaptosomes. Although studies in yeast revealed that KCC2 is selected for endoplasmic reticulum-associated degradation (ERAD), experiments in HEK293 cells supported a more subtle role for ERAD in maintaining steady-state levels of KCC2. Nevertheless, this system allowed for an analysis of KCC2 glycosylation in the ER and Golgi, which serves as a read-out for transport through the secretory pathway. In turn, KCC2 was remarkably stable in primary rat neurons, suggesting that KCC2 folds efficiently in more native systems. Consistent with these data, the mature glycosylated form of KCC2 was abundant in primary rat neurons as well as in rat and human brain. Together, this work details the first insights into the influence that the cellular and membrane environments have on several fundamental KCC2 properties, acknowledges the advantages and disadvantages of each system, and helps set the stage for future experiments to assess KCC2 in a normal or disease setting.

Clobenpropit, a histamine H3 receptor antagonist/inverse agonist, inhibits [3H]-dopamine uptake by human neuroblastoma SH-SY5Y cells and rat brain synaptosomes.

Clobenpropit, a potent antagonist/inverse agonist at the histamine H3 receptor (H3R), reduced the cytotoxic action of 6-hydroxydopamine (6-OHDA) in neuroblastoma SH-SY5Y cells transfected with the human H3R. We therefore set out to study whether this effect involved a receptor-independent action on dopamine transport. The uptake of [3H]-dopamine was assayed in SH-SY5Y cells and rat striatal or cerebro-cortical isolated nerve terminals (synaptosomes). Clobenpropit binding to the human norepinephrine (NET) and dopamine (DAT) transporters was analyzed by molecular modeling. In SH-SY5Y cells, [3H]-dopamine uptake was inhibited by desipramine (selective NET inhibitor), GBR-12909 (selective DAT inhibitor), and fluoxetine (selective inhibitor of the serotonin transporter, SERT) with IC50 values 37, 537, and 2800nM, respectively. The potency rank order indicates that [3H]-dopamine uptake is primarily performed by NET. Clobenpropit inhibited [3H]-dopamine uptake (maximum inhibition 82.7±2.8%, IC50 490nM), and the effect was reproduced by the H3R antagonist/inverse agonist iodophenpropit, but not by the agonists R-α-methylhistamine and immepip or the antagonists/inverse agonists ciproxifan and A-331440. Clobenpropit also inhibited [3H]-dopamine uptake by rat striatal and cerebro-cortical synaptosomes (-54.6±11.3% and -46.3±9.6%, respectively, at 10μM). Modeling of the human NET and DAT obtained by homology from the crystal of Drosophila melanogaster DAT showed that clobenpropit can bind to a site also recognized in both transporters by nisoxetine, a potent NET inhibitor. These data indicate a direct inhibitory effect of clobenpropit on catecholamine transport.

REGULATED RELEASE OF HYPER-PHOSPHORYLATED, AGGREGATED AND SEEDING COMPETENT TAU FROM RODENT BRAINS AND HUMAN SYNAPTOSOMES

Tau is a microtubule binding protein expressed in neuronal axons and nerve terminals. Mutations in tau are known to cause different neurological diseases and tau pathology, in the form of aggregates and tangles, is a hallmark of Alzheimer's disease. Tau pathology in AD starts in the entorhinal cortex but “spreads” along synaptically connected areas as the disease progresses. Understanding how tau is trafficked to the nerve terminal, secreted, and taken up by postsynaptic neurons could lead to the identification of novel mechanisms aimed at halting disease progression in AD. Synaptic structures (synaptosomes) were purified from the brains of transgenic mice harbouring pathological human tau (P301S and tg4510) and from human control and AD subjects. Western blots were used to detect synaptic proteins and tau. STED microscopy was used to evalaute tau sub-cellular localisation. The ability of synaptic tau to “seed” pathological tau aggregation was evaluated in transfected HEK293 and rat cortical neurons. Calcium dependent release was induced by KCl or ionomycin, while botulinum toxins and pharmacological agents were used to cleave SNARE proteins and block exocytosis. Pathologically hyper-phosphorylated (AT8+) aggregated (Sarcosyl-insoluble) and seeding-competent tau was found in synaptosomes from both transgenic mice and human AD brains. Pathological tau was enriched in synaptosomes of the most affected brain areas in the mice, while in human brain synaptosomes the levels of pathological tau were correlated with the patient's Braak stage. STED analysis demonstrated that pathological tau was enriched in the presynaptic compartment. Pathological tau was released in a calcium dependent manner by both KCl and ionomycin. Botulinum toxin A and D were found to cleave synaptosomal SNAP25 and synaptobrevin, respectively, and at the same time inhibit calcium-dependent tau release. All together these data build on previous evidence of pathological tau presence in presynaptic terminals and demonstrate its calcium-dependent release. Pathological tau release requires intact SNARE proteins, suggesting a vesicular, exocytotic, process. This could represent a critical step in the spreading of tau in AD brain. Therapeutic interventions aimed at blocking tau release could be beneficial in reducing tau propagation and disease progression in AD.

The Human Immunodeficiency Virus-1 Protein Transactivator of Transcription Up-RegulatesN-Methyl-d-aspartate Receptor Function by Acting at Metabotropic Glutamate Receptor 1 Receptors Coexisting on Human and Rat Brain Noradrenergic Neurones

We investigated the effects of the human immunodeficiency virus-1 transactivator of transcription (Tat) on the release of norepinephrine (NE) from human and rat brain synaptosomes. Tat could not evoke directly release of [3H]NE. In the presence of Tat (1 nM), N-methyl-D-aspartate (NMDA) concentrations unable to release (human synaptosomes) or slightly releasing (rat synaptosomes) [3H]NE became very effective. The NMDA/Tat-evoked release depends on NMDA receptors (NMDARs) since it was abolished by MK-801 (dizocilpine). Tat binding at NMDARs was excluded. The NMDA-induced release of [3H]NE in the presence of glycine was further potentiated by Tat. The release evoked by NMDA/glycine/Tat depends on metabotropic glutamate receptor 1 (mGluR1) activation, since it was halved by mGluR1 antagonists. Tat seems to act at the glutamate recognition site of mGluR1. Recently, Tat was shown to release [3H]acetylcholine from human cholinergic terminals; here, we demonstrate that this effect is also mediated by presynaptic mGluR1. The peptide sequence Tat41-60, but not Tat61-80, mimicked Tat. Phospholipase C, protein kinase C, and cytosolic tyrosine kinase are involved in the NMDA/glycine/Tat-evoked [3H]NE release. To conclude, Tat can represent a potent pathological agonist at mGlu1 receptors able to release acetylcholine from human cholinergic terminals and up-regulate NMDARs mediating NE release from human and rat noradrenergic terminals.

No modulatory effect of neurokinin-1 receptor antagonists on serotonin uptake in human and rat brain synaptosomes

Some studies have demonstrated antidepressant activity of neurokinin-1-receptor antagonists (NK-1-RA) in major depressive disorder. However, the underlying mechanisms of this antidepressant effect are largely unknown. Preclinical studies in rats and mice have suggested that NK-1-RA do increase the neuronal release of serotonin (5-HT). This, however, seems to be compensated by an increased 5-HT reuptake, indicating that NK-1-RA have no inhibitory effect on the 5-HT transporter in rodents. Given the possibility that modulation of neurotransmitter release and reuptake may differ between species, with major differences found between rodents and humans, we investigated for the first time the possible modulatory effect of NK-1-RA on 5-HT uptake in human brain synaptosomes and compared it with the situation in rat cortex. We found that the specific human NK-1-RA L-733060, in contrast to the SSRI fluvoxamine (IC 50 = 10 − 7.96 M) did not inhibit 5-HT uptake in human brain synaptosomes and did not modulate fluvoxamine-induced 5-HT uptake inhibition at 1 μM. Furthermore, substance P as well as Sar 9Met(O 2) 11SP, as the major agonists at the NK-1-R, did not modulate 5-HT uptake in human brain synaptosomes. Similar results were found in rat cortex synaptosomes by using the rat-specific NK-1-RA WIN51708. These results show that in humans, as in rodents, inhibition of the 5-HT transporter is probably not the underlying mechanism of the assumed antidepressant activity of NK-1-RA.

Serotonin transport kinetics correlated between human platelets and brain synaptosomes

Blood platelets have been used extensively as a model system for investigating the role of the serotonin transporter (SERT) in various psychiatric disorders, especially depression. However, to date, it is not known whether platelet serotonin (5-HT) transport would be related to that in brain. We examined 5-HT transport kinetics simultaneously in human blood platelets and human cortical brain synaptosomes to determine whether they were correlated. Blood platelets and synaptosomes were obtained from 25 patients undergoing epileptic surgery. Synaptosomes were obtained from normal margins of surgical neuropathology specimens of anterotemporal cortex. Platelet SERT V(max) was significantly correlated with brain SERT V(max) on linear regression (r=0.58, p<0.005), after controlling for the confounding effects of gender (t=-2.4, p=0.025) and time of day (t=2.1, p<0.05). Consistent with previous observations, there was a negative correlation between the maximum velocity (V(max)) of platelet 5-HT transport and pO2 (r=-0.52, p<0.01). Females had a significantly higher pO2 than males (F=4.9, p<0.05). After accounting for gender differences, addition of pO2 did not add further strength to the regression, given the aforementioned gender differences in pO2. The correlation between unadjusted values for platelet vs brain SERT V(max) was r=0.3, p=0.06. These results suggest that a relationship may exist between 5-HT transport in platelets and cortical synaptosomes, when appropriate controls for confounding factors are employed.

Lamotrigine inhibits monoamine uptake in vitro and modulates 5-hydroxytryptamine uptake in rats

Lamotrigine is a novel anticonvulsant drug which also stabilises mood in bipolar illness via an unknown mechanism. We report the concentration-dependent inhibition of 5-hydroxytryptamine (5-HT) uptake in both human platelets and rat brain synaptosomes (IC 50s were 240 and 474 μM, respectively) by lamotrigine. Synaptosomal uptake of noradrenaline (IC 50 239 μM) and dopamine (IC 50 322 μM) was also inhibited. Tetrodotoxin failed to modulate 5-HT uptake suggesting that sodium channel blockade does not mediate the lamotrigine effect. Lithium, sodium valproate, zonisamide, and carbamazepine all possess anti-manic activity but only the latter inhibited 5-HT uptake. The inhibition of the p-chloroamphetamine-induced 5-HT syndrome in rats suggests that lamotrigine also inhibits 5-HT uptake in vivo. These effects probably reflect an affinity for biogenic amine transporters. However, at present, it remains uncertain whether, at clinically effective doses, these effects contribute significantly to the efficacy of lamotrigine in bipolar illness.

No evidence for specific opioid effects on batrachotoxin-modified sodium channels from human brain synaptosomes

Human central nervous system (CNS) sodium channels modified by batrachotoxin and incorporated inter voltage-clamped lipid bilayers, were exposed to various concentrations of the opioid alfentanil (0.2–8.0 mM). Alfentanil caused a concentration-dependent and membrane potential independent reduction of the single channel amplitude and the fractional channel open-time. The weighted computer fit of the dose-response curve yielded a maximal conductance block of 50% with an EC 50 of 1.3 mM. These effects occurred at levels beyond clinically relevant human serum/brain levels (0.003 mM) but within the predicted concentration range using the Meyer–Overton (lipid solubility/anaesthetic potency) correlation. Thus, human CNS sodium channels are probably not a main target site for the clinical effects of alfentanil but they provide a model system to estimate the proportion of the lipophilic interactions contributing to its overall effect.

Human brain N-methyl-D-aspartate receptors regulating noradrenaline release are positively modulated by HIV-1 coat protein gp120.

To investigate the effect of HIV-1 gp120 on the function of glutamate receptors of the N-methyl-D-aspartate (NMDA) type in the human brain. The monitoring of neurotransmitter release from superfused isolated nerve endings is widely recognized as a technique appropriate for the study of neurotransmitter release and to attribute a precise localization to the site(s) of action of drugs able to modulate release. Synaptosomes (pinched-off nerve endings) were prepared from fresh human brain tissue samples removed during neurosurgery, labelled with [3H]-noradrenaline and superfused at a rate of 0.5 ml/min with NMDA in the presence of gp41, gp160, gp120 or the V3 loop, with or without NMDA receptor antagonists. Fractions of superfusate were collected and measured for radioactivity. NMDA elicited a glycine-sensitive release of [3H]-noradrenaline from human brain synaptosomes. HIV-1 gp120 potentiated the NMDA (1 mM)-evoked [3H]-noradrenaline release (maximal effect approximately 110% at 1 nM). The release elicited by NMDA plus gp120 was prevented by the classical NMDA receptor antagonists dizocilpine or 7-chlorokynurenic acid, as well as by memantine. The potentiation by gp120 of the NMDA-evoked [3H]-noradrenaline release was mimicked by gp160 but not by gp41. The effect of gp120 was retained by the V3 loop. Finally, gp120 reversed (1 nM) and surmounted (10nM) the antagonism by 10 microM 7-chlorokynurenate of the NMDA-evoked [3H]-noradrenaline release. gp 120 binds directly through the V3 loop at noradrenergic axon terminals in human brain neocortex and may alter the function of presynaptic NMDA receptors mediating regulation of noradrenaline release.

Effect of plasma from premenstrual syndrome and control patients on human platelet and rat brain synaptosome monoamine oxidase B activity.

The present study determined the kinetic properties of platelet monoamine oxidase (MAO) in patients with premenstrual syndrome (PMS) and control subjects during the pre- and postmenstrual intervals. In addition, we investigated the effects of plasma obtained during the pre- and postmenstrual intervals from both subject groups on MAO activity in washed human platelets and rat brain synaptosomes. The Vmax of platelet MAO in postmenstrual PMS patients was significantly less than that during the premenstrual phase. Plasma from both subject groups (10-100 microliters) inhibited platelet and synaptosomal MAO in a dose-dependent manner to approximately the same degree at each time interval. The results indicate that although human plasma contains endogenous substances which inhibit MAO activity, alterations in their concentration are probably not responsible for the previously reported transient changes in platelet MAO activity in PMS.

3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and other heterocyclic amines as inhibitors of mitochondrial monoamine oxidases separated from human brain synaptosomes

Effects of a series of heterocyclic amines on type A and B monoamine oxidases were studied using human brain synaptosomal mitochondria as an enzyme source. 3-Amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) was the most potent inhibitor of the heterocyclic amines examined. It inhibited type A monoamine oxidase in competition with the substrate and the K(i) value toward type A was 0.84 +/- 0.06 ?M, which was about 1/50 of its K(m) value toward an amine substrate, kynuramine; 46.4 +/- 11.4 ?M. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was also a potent inhibitor. 3- or 2-Aminopyridoindoles or -imidazoles inhibited both type A and B monoamine oxidase competitively to substrate, but inhibition of type B monoamine oxidase by amines with 2-amino-3-methylimidazol ring was non-competitive. In general, type A monoamine oxidase was more sensitive to these heterocyclic amines than type B; the K(i) values toward type A were much smaller than those of type B. Inhibition of type A monoamine oxidase by Trp-P-2 proved to be reversible; the enzyme activity inhibited by Trp-P-2 could be fully recovered by removal of Trp-P-2 by column chromatography with Sephadex G-25.

Synthesis, structure, and action of some gossypol derivatives on the peroxidation of the lipids of biosubstrates

The synthesis has been effected of new gossypol derivatives using piperidino-and morpholinoethylamines. According to their PMR spectra, these substances exist in the keto-amine form. Their action on the peroxidation of lipids (POL) of various biosubstrates has been studied. Gossypol bis(piperinoethylimine) and bis(morpholinoethylimine) in concentrations of 1·10−7–5·10−6 mM exert a pronounced antioxidant action on human blood serum and rat brain synaptosomes. In the same concentrations, these substances suppressed the POL in enzymatic and nonenzymatic systems of the oxidation of rat liver microsomes.

Uptake of glycosylated β-galactosidase into human brain synaptosomes: Occurrence of two different systems for specific glycosides

Uptake of glycoproteins into brain nerve terminals was studied, using glycosylated β-galactosidase and human brain synaptosomes. A series of p-aminophenyl α- or β-glycosides were covalently bound to β-galactosidase purified from Aspergillus oryzae and uptake of the enzyme into human brain synaptosomes was measured. β-Galactosidase coupled with α- d-mannoside (α- d-Man β-gal) was the best to be taken up among glycoconjugates tested. The uptake was dependent on the amount of α- d-Man β-gal, the incubation temperature and the incubation time. Kinetic studies showed that the uptake was a saturable and receptor-mediated process, which followed Michaelis-Menten kinetics. The specificity of the uptake was confirmed by competition of the uptake of α- d-Man β-gal by α-methyl mannoside. The uptake was dependent on Ca 2+ and that of β-galactosidase coupled with α- l-fucoside (α- l-Fuc β-gal) was saturated by Ca 2+ at lower concentrations than that Of α- d-Man β-gal.Occurrence of two different uptake systems was also suggested by kinetic study; a low-affinity and high-capacity system for α- d-mannoside and a high-affinity and low-capacity system for α- l-fucoside. The former system may be mediated by a process coupled with a Ca 2+/calmodulin-dependent protein kinase, but the later may not be dependent on the protein kinase.

Studies in postmortem dopamine uptake. I. Kinetic characterization of the synaptosomal dopamine uptake in rat and human brain after postmortem storage and cryopreservation. Comparison with noradrenaline and serotonin uptake.

The effect of various post-mortem storage times and temperatures on the kinetic parameters of synaptosomal high-affinity dopamine (DA) uptake was studied in rat nucleus accumbens. After post-mortem storage up to 48 hours of rat heads in situ at 22 degrees C the KM data increase moderately in contrast to the Vmax data decreasing rapidly (t1/2 = 30 hours); at 2 degrees C similar changes were observed. The post-mortem changes of the kinetic parameters of noradrenaline (NA) and serotonin (5 HT) uptake were shown in this model to be similar. It is proposed to use this animal model for correcting the kinetic data of DA, NA and 5 HT uptake in human brains regarding the various post-mortem delays. Furthermore, in cryopreservation experiments using a two-step freezing procedure in liquid nitrogen of brain pieces preincubated with dimethylsulfoxide DA uptake of rat and human brain synaptosomes was unchanged after freezing-thawing. The same is true for both NA and 5 HT uptake as demonstrated in human brain tissue. Therefore, post-mortem human brain seems to be suitable for investigating synaptosomal DA, NA, and 5 HT uptake after cryopreservation and correcting for post-mortem delay, when determining kinetic parameters.

Binding of specific glycoconjugates to human brain synaptosomes: studies using glycosylated β-galactosidase

Using glycosylated β-galactosidase (β-gal) as a glycoprotein model, binding of glycoconjugates to human brain synaptosomes was studied. Out of β-gal modified with a series of p-aminophenyl α- or β-glycosides, β-gal modified with p-aminophenyl β- d-glucopyranoside (β- d-Glc β-gal) was bound to the synaptosomes most effectively, then β-gal modified with β- d-galactoside and with α- d-mannoside. Kinetic studies on the binding of β- d-Glc β-gal indicated the presence of saturable binding on human brain synaptosomes. The values of the apparent K m and of the maximal binding velocity were obtained to be 248 ± 32.9 μM and 43.8 ± 1.43 fmol/min/mg synaptosome protein, at 4°C and pH 7.5. The specificity of the sugar recognition site proved by the competitive inhibition of the binding of β- d-Glc β-gal by bovine serum albumin modified with the same glycoside. The binding of β-gal modified with β- d-galactoside was inhibited by treatment of the synaptosomes with trypsin, phospholipase A 2, C and D, and with neuraminidase, while the binding of β- d-Glc β-gal was inhibited by neuraminidase treatment of synaptosomes. In subcellular fractions of human brain the binding protein was localized mainly in synaptosomes.

1,4-benzoquinone as a new inhibitor of monoamine oxidase

The effect of 1,4-benzoquinone (BQ) on type A and B monoamine oxidase (MAO-A and -B) in human brain synaptosomes was examined. MAO-A was found to be markedly inhibited by BQ competitively with the substrate, kynuramine, while MAO-B was less sensitive to this inhibitor and the inhibition was noncompetitive with the substrate. The K i value of MAO-A (9.62±0.35 μM) was much smaller than the K m value of the enzyme with the substrate (56.3±3.5 μM) or the K i value of MAO-B with BQ (20.3±0.9 μM). The inhibition of MAO-A by BQ was also confirmed by the use of placental mitochondria and clonal rat pheochromocytoma PC12h cells as enzyme sources. The inhibition of MAO activity by BQ proved to be reversible: the inhibited enzyme activity could be recovered by column chromatography on Sephadex G-25.

Uptake of L-threo-dihydroxyphenylserine into human brain synaptosomes.

Out of four diastereoisomers of 3,4-dihydroxyphenylserine (DOPS), L-threo- and L-erythro-isomer were found to be taken up into human brain synaptosomes. The uptake of L-threo-DOPS was dependent on the temperature and sensitive to the metabolic inhibitors. The L-threo-DOPS uptake proved to be saturable and carrier-mediated transport with two different kinetic characteristics; a high-affinity and low-capacity and a low-affinity and high-capacity system. The apparent Km values of these two systems were obtained to be 28.6 microM and 2.47 mM, respectively. The high-affinity transport was inhibited by glycine, L-tyrosine, L-proline, L-serine, L-Dopa, L-tryptophan, and L-phenylalanine. The inhibition by L-tyrosine was competitive in regard to L-threo-DOPS. The L-threo-DOPS uptake was inhibited by 2,4-dinitrophenol, sodium cyanide and other uncouplers of oxidative phosphorylation and by ouabain, an inhibitor of Na+, K+-ATPase, indicating that the uptake is coupled to ATP hydrolysis. On the other hand, L-threo-DOPS uptake by the low-affinity system was not inhibited by metabolic inhibitors, indicating that it may be facilitated diffusion common to high concentrations of L-amino acids.

Intracellular transport in neurons.

Intracellular transport in neurons.

Fatty acid composition of lipids of human brain myelin and synaptosomes: Changes in phenylketonuria and down's syndrome

1. 1. The fatty acid composition of phosphoglycerides and sphingolipids in myelin and phosphoglycerides in synaptosomes isolated from brain tissue of patients with phenylketonuria, and Down's Syndrome is compared with the fatty acid composition of lipids in myelin and synaptosomes from normal human brain. 2. 2. The implications of changes in the fatty acid composition of lipids in myelin and synaptosomes from PKU and DS brains with respect to functional abnormality of the membranes are discussed.