Synaptic Vesicle Preparations Research Articles

The Synaptosome as a Model System for Studying Synaptic Physiology

Alongside rodent brain slices and primary neuronal cultures, synaptosomes (isolated nerve terminals) have been an important model system for studying the molecular mechanisms of synaptic function in the brain. Synaptosomes were first prepared in the late 1950s by Whittaker and colleagues and were instrumental in studying synaptic structure and defining the functional components of the synapse, including the identity of the major neurotransmitters and their uptake mechanisms. Synaptosomes can also be stimulated to release neurotransmitters and were used to discover a number of regulatory signaling pathways that fine-tune synaptic transmission. In the past decade, landmark proteomic studies of synaptosomes and synaptic vesicle preparations have further dissected the protein composition of the synapse. This introduction briefly describes the history of the synaptosome preparation and highlights how it continues to be relevant as our focus in the neuroscience community centers on synaptic dysfunction in aging and neurological disease.

Subcellular fractionation of the brain: preparation of synaptosomes and synaptic vesicles.

The human brain is estimated to contain trillions of synaptic nerve terminals. These are the connections between neurons that are responsible for transmitting information and are modified as a result of learning. A valuable tool for studying synapses is the isolated nerve terminal, or synaptosome, which is obtained by homogenizing the brain in such a way that individual synapses pinch off to form metabolically active compartments that can recapitulate neurotransmitter release. This protocol describes the stepwise fractionation of rat brain tissue to yield synaptosomes and synaptic vesicles, which can be used in many different experimental approaches to study the structure and protein composition of the synapse and even dissect the molecular mechanisms of neurotransmission.

The profile of mephedrone on human monoamine transporters differs from 3,4-methylenedioxymethamphetamine primarily by lower potency at the vesicular monoamine transporter

Mephedrone (4-methylmethcathinone, MMC) and 3,4-methylenedioxymethamphetamine (MDMA) are constituents of popular party drugs with psychoactive effects. Structurally they are amphetamine-like substances with monoamine neurotransmitter enhancing actions. We therefore compared their effects on the human monoamine transporters using human cell lines stably expressing the human noradrenaline, dopamine and serotonin transporter (NET, DAT and SERT); preparations of synaptic vesicles from human striatum in uptake experiments; and a superfusion system where releasing effects can be reliably measured. MMC and MDMA were equally potent in inhibiting noradrenaline uptake at NET, with IC50 values of 1.9 and 2.1µM, respectively. Compared to their NET inhibition potency, both drugs were weaker uptake inhibitors at DAT and SERT, with MMC being more potent than MDMA at DAT (IC50: 5.9 vs 12.6µM) and less potent than MDMA at SERT (IC50: 19.3 vs 7.6µM). MMC and MDMA both induced concentration-dependently [3H]1-methyl-4-phenylpyridinium-release from NET-, DAT or SERT-expressing cells which was clearly transporter-mediated release as demonstrated by the selective inhibitory effects of nmolar to low µmolar concentrations of desipramine, GBR 12909 and fluoxetine, respectively. MMC and MDMA differed most in their inhibition of [3H]dopamine uptake by synaptic vesicles from human striatum with MDMA being 10-fold more potent than MMC (IC50: 20 vs 223µM) and their ability to release [3H]dopamine from human vesicular monoamine transporter expressing SH-SY5Y neuroblastoma cells in which MDMA seems to have a stronger effect. Our findings give a molecular explanation to the lower long-term neurotoxicity of MMC compared to MDMA.

SLC10A4 Is a Vesicular Amine-Associated Transporter Modulating Dopamine Homeostasis

BackgroundThe neuromodulatory transmitters, biogenic amines, have profound effects on multiple neurons and are essential for normal behavior and mental health. Here we report that the orphan transporter SLC10A4, which in the brain is exclusively expressed in presynaptic vesicles of monoaminergic and cholinergic neurons, has a regulatory role in dopamine homeostasis. MethodsWe used a combination of molecular and behavioral analyses, pharmacology, and in vivo amperometry to assess the role of SLC10A4 in dopamine-regulated behaviors. ResultsWe show that SLC10A4 is localized on the same synaptic vesicles as either vesicular acetylcholine transporter or vesicular monoamine transporter 2. We did not find evidence for direct transport of dopamine by SLC10A4; however, synaptic vesicle preparations lacking SLC10A4 showed decreased dopamine vesicular uptake efficiency. Furthermore, we observed an increased acidification in synaptic vesicles isolated from mice overexpressing SLC10A4. Loss of SLC10A4 in mice resulted in reduced striatal serotonin, noradrenaline, and dopamine concentrations and a significantly higher dopamine turnover ratio. Absence of SLC10A4 led to slower dopamine clearance rates in vivo, which resulted in accumulation of extracellular dopamine. Finally, whereas SLC10A4 null mutant mice were slightly hypoactive, they displayed hypersensitivity to administration of amphetamine and tranylcypromine. ConclusionsOur results demonstrate that SLC10A4 is a vesicular monoaminergic and cholinergic associated transporter that is important for dopamine homeostasis and neuromodulation in vivo. The discovery of SLC10A4 and its role in dopaminergic signaling reveals a novel mechanism for neuromodulation and represents an unexplored target for the treatment of neurological and mental disorders.

Activity-independent release of the amyloid β-peptide from rat brain nerve terminals

Synaptic degeneration is one of the earliest hallmarks of Alzheimer disease. The molecular mechanism underlying this degeneration is not fully elucidated but one key player appears to be the synaptotoxic amyloid β-peptide (Aβ). The exact localization of the production of Aβ and the mechanisms whereby Aβ is released remain elusive. We have earlier shown that Aβ can be produced in crude synaptic vesicle fractions and it has been reported that increased synaptic activity results in increased secreted but decreased intracellular Aβ levels. Therefore, we considered whether Aβ could be produced in synaptic vesicles and/or released through the same mechanisms as neurotransmitters in synaptic vesicle exocytosis. Small amounts of Aβ were found to be produced in pure synaptic vesicle preparations. We also studied the release of glutamate and Aβ from rat cortical nerve terminals (synaptosomes). We found that large amounts of Aβ were secreted from non-stimulated synaptosomes, from which glutamate was not released. On the contrary, we could not detect any differences in Aβ release between non-stimulated synaptosomes and synaptosomes stimulated with KCl or 4-aminopyridine, whereas glutamate release was readily inducible in this system. To conclude, our results indicate that the major release mechanism of Aβ from isolated nerve terminals differs from the synaptic release of glutamate and that the activity-dependent increase of secreted Aβ, reported by several groups using intact cells, is likely dependent on post-synaptic events, trafficking and/or protein synthesis mechanisms.

Vessel wall characteristics using high-resolution magnetic resonance imaging in reversible cerebral vasoconstriction syndrome and central nervous system vasculitis

Synaptic degeneration is one of the earliest hallmarks of Alzheimer disease. The molecular mechanism underlying this degeneration is not fully elucidated but one key player appears to be the synaptotoxic amyloid β-peptide (Aβ). The exact localization of the production of Aβ and the mechanisms whereby Aβ is released remain elusive. We have earlier shown that Aβ can be produced in crude synaptic vesicle fractions and it has been reported that increased synaptic activity results in increased secreted but decreased intracellular Aβ levels. Therefore, we considered whether Aβ could be produced in synaptic vesicles and/or released through the same mechanisms as neurotransmitters in synaptic vesicle exocytosis. Small amounts of Aβ were found to be produced in pure synaptic vesicle preparations. We also studied the release of glutamate and Aβ from rat cortical nerve terminals (synaptosomes). We found that large amounts of Aβ were secreted from non-stimulated synaptosomes, from which glutamate was not released. On the contrary, we could not detect any differences in Aβ release between non-stimulated synaptosomes and synaptosomes stimulated with KCl or 4-aminopyridine, whereas glutamate release was readily inducible in this system. To conclude, our results indicate that the major release mechanism of Aβ from isolated nerve terminals differs from the synaptic release of glutamate and that the activity-dependent increase of secreted Aβ, reported by several groups using intact cells, is likely dependent on post-synaptic events, trafficking and/or protein synthesis mechanisms.

Characterization of presynaptic septin complexes in mammalian hippocampal neurons

Septins are GTPases that form heteromeric complexes and are linked to neurological disorders. Although several septin subcomplexes have been reported in various mammalian tissues, the cellular and subcellular distribution of these complexes is largely unexplored. Using antibodies against ten mammalian septins, we show that septins diverge with respect to their tissue distribution implying that septin complexes in various tissues have unique composition. Although all ten septins examined were expressed in brain tissue, we describe septin complex(es) including SEPT3, SEPT5, SEPT6, SEPT7 and SEPT11 that could be functional within the presynapse because, unlike other septins they: (1) showed an increase in expression from embryonic day 15 to post-natal day 70, (2) were abundantly expressed in axons and growth cones of developing hippocampal neurons, (3) were found in presynaptic terminals of mature synapses, (4) were enriched in a preparation of synaptic vesicles and (5) immunoprecipitated together from brain tissue and cultured nerve cells. Knockdown of SEPT5 or SEPT7 in developing hippocampal neurons impaired axon growth. Because septins are functionally linked to the cytoskeleton and vesicle traffic, presynaptic neuronal septin complexes could be important for ensuring proper axon development and neurotransmitter release.

The Novel Pyrrolidine Nor-Lobelane Analog UKCP-110 [cis-2,5-di-(2-phenethyl)-pyrrolidine hydrochloride] Inhibits VMAT2 Function, Methamphetamine-Evoked Dopamine Release, and Methamphetamine Self-Administration in Rats

Both lobeline and lobelane attenuate methamphetamine self-administration in rats by decreasing methamphetamine-induced dopamine release via interaction with vesicular monoamine transporter-2 (VMAT2). A novel derivative of nor-lobelane, cis-2,5-di-(2-phenethyl)-pyrrolidine hydrochloride (UKCP-110), and its trans-isomers, (2R,5R)-trans-di-(2-phenethyl)-pyrrolidine hydrochloride (UKCP-111) and (2S,5S)-trans-di-(2-phenethyl)-pyrrolidine hydrochloride (UKCP-112), were evaluated for inhibition of [(3)H]dihydrotetrabenazine binding and [(3)H]dopamine uptake by using a rat synaptic vesicle preparation to assess VMAT2 interaction. Compounds were evaluated for inhibition of [(3)H]nicotine and [(3)H]methyllycaconitine binding to assess interaction with the major nicotinic receptor subtypes. In addition, compounds were evaluated for inhibition of methamphetamine-evoked endogenous dopamine release by using striatal slices. The most promising compound, UKCP-110, was evaluated for its ability to decrease methamphetamine self-administration and methamphetamine discriminative stimulus cues and for its effect on food-maintained operant responding. UKCP-110, UKCP-111, and UKCP-112 inhibited [(3)H]dihydrotetrabenazine binding (K(i) = 2.66 ± 0.37, 1.05 ± 0.10, and 3.80 ± 0.31 μM, respectively) and had high potency inhibiting [(3)H]dopamine uptake (K(i) = 0.028 ± 0.001, 0.046 ± 0.008, 0.043 ± 0.004 μM, respectively), but lacked affinity at nicotinic receptors. Although the trans-isomers did not alter methamphetamine-evoked dopamine release, UKCP-110 inhibited (IC(50) = 1.8 ± 0.2 μM; I(max) = 67.18 ± 6.11 μM) methamphetamine-evoked dopamine release. At high concentrations, UKCP-110 also increased extracellular dihydroxyphenylacetic acid. It is noteworthy that UKCP-110 decreased the number of methamphetamine self-infusions, while having no effect on food-reinforced behavior or the methamphetamine stimulus cue. Thus, UKCP-110 represents a new lead in the development of novel pharmacotherapies for the treatment of methamphetamine abuse.

Alpha-synuclein is localized in a subpopulation of rat brain synaptic vesicles.

Alpha-synuclein is a neuronal protein implicated both in synaptic transmission and in neurodegenerative diseases. Although it is evident that this protein is enriched in the presynaptic terminals of neurons, localization in synaptic vesicles has not been conclusively determined. Here, we show that alpha-synuclein is present, but not enriched, in synaptic vesicles using highly purified synaptic vesicle preparations from rat brain homogenate. Immunoisolation of vesicles using antibodies against synaptophysin or synaptobrevin confirmed the presence of alpha-synuclein in synaptic vesicles. Additional separation of synaptic vesicles by sucrose velocity centrifugation showed that there are different subpopulations of synaptic vesicles and that alpha-synuclein is present only in a specific subpopulation, whereas synaptophysin and synaptobrevin were found in all the synaptic vesicles. Presence of alpha-synuclein only in a subset of synaptic vesicles suggests that this protein may have a specific function in synaptic vesicle cycling, hence in synaptic transmission.

The First Luminal Domain of Vesicular Monoamine Transporters Mediates G-protein-dependent Regulation of Transmitter Uptake

The activity of vesicular monoamine transporters (VMATs) is down-regulated by the G-protein alpha-subunits of G(o2) and G(q), but the signaling pathways are not known. We show here that no such regulation is observed when VMAT1 or VMAT2 are expressed in Chinese hamster ovary (CHO) cells. However, when the intracellular compartments of VMAT-expressing CHO cells are preloaded with different monoamines, transport becomes susceptible to G-protein-dependent regulation, with differences between the two transporter isoforms. Epinephrine induces G-protein-mediated inhibition of transmitter uptake in CHOVMAT1 cells but prevents inhibition induced by dopamine in CHOVMAT2 cells. Epinephrine also antagonizes G-protein-mediated inhibition of monoamine uptake by VMAT2 expressing platelets or synaptic vesicles. In CHOVMAT2 cells G-protein-mediated inhibition of monoamine uptake can be induced by 5-hydroxytryptamine (serotonin) 1B receptor agonists, whereas alpha1 receptor agonists modulate uptake into CHOVMAT1 cells. Accordingly, 5-hydroxytryptamine 1B receptor antagonists prevent G-protein-mediated inhibition of uptake in partially filled platelets and synaptic vesicles expressing VMAT2. CHO cells expressing VMAT mutants with a shortened first vesicular loop transport monoamines. However, no or a reduced G-protein regulation of uptake can be initiated. In conclusion, vesicular content is involved in the activation of vesicle associated G-proteins via a structure sensing the luminal monoamine content. The first luminal loop of VMATs may represent a G-protein-coupled receptor that adapts vesicular filling.

Characterization of the localization and function of NECAP 1 in neurons

NECAPs (adaptin ear-binding clathrin-associated protein) are a new family of clathrin accessory proteins identified through a proteomic analysis of clathrin-coated vesicles (CCVs) from the brain. One member of this family, NECAP 1, is found primarily in tissues from the central nervous system and has been shown to be complexed tightly with a substantial portion of adaptor protein-2 (AP-2) in brain extracts. However, the function and intracellular location of this protein is unknown. In this study, we find that endogenous and epitope-tagged NECAP 1 co-localizes well with clathrin and AP-2 in punctate structures, many of which also contain the presynaptic markers synaptophysin, synaptotagmin or synaptic vesicle protein 2 (SV2). NECAP 1 was also detected by western blot in synaptic vesicle preparations. Overexpression of a truncation mutant of NECAP 1 (BC-NECAP 1) in neurons inhibited transferrin endocytosis but not epidermal growth factor (EGF) endocytosis, and this inhibition was dependent on an AP-2-binding WVQF motif. Moreover, overexpression of BC-NECAP 1 results in inhibition of synaptotagmin endocytosis both in unstimulated neurons and in neurons stimulated with potassium chloride. This inhibition was abrogated by truncation of the WVQF domain. We conclude from these observations that NECAP 1 plays a role in clathrin-mediated neuronal endocytosis, including a role in presynaptic endocytosis.

In Vitro Assessment of Dopamine Uptake and Methamphetamine‐Induced Dopamine Efflux at the Vesicular Monoamine Transporter‐2

The vesicular monoamine transporter-2 (VMAT-2) is a vesicular transporter protein that utilizes proton electrochemical gradients to sequester cytoplasmic dopamine (DA) into vesicles for storage and subsequent release. Herein, we report the first use of rotating disk electrode (RDE) voltammetry to measure DA uptake and efflux by the VMAT-2 in small synaptic vesicles. DA uptake profiles followed mixed order kinetics with apparent zero order kinetics for the first 25 s and apparent first order kinetics thereafter. Vesicular DA uptake was temperature- and ATP-dependent and was blocked by the VMAT-2 inhibitor tetrabenazine. Initial velocities of DA uptake followed Michaelis-Menten kinetics with a Km and Vmax of 289 ± 59 nM and 1.9 ± 0.2 fmol / (s * μg protein), respectively. The VMAT-2 substrate analog, methamphetamine, caused DA efflux while the VMAT-2 inhibitor, tetrabenazine, did not. Methamphetamine-induced DA efflux had an initial velocity of 0.54 ± 0.08 fmol / (s * μg protein) and was blocked by tetrabenazine. These results suggest that RDE voltammetric measurements are selective for VMAT-2 uptake activity in preparations of small synaptic vesicles and will allow the design of experiments that could reveal important information about the kinetics of VMAT-2 activity and its inhibition. Additionally, methamphetamine-induced DA efflux in these preparations is not diffusive but rather is mediated by the VMAT-2. This work was supported by grants DA 00689, DA 04222, DA 13367, DA 11389, DA 019447, and DA 00378 from the National Institute on Drug Abuse.

A Combinatorial Code for the Interaction of α-Synuclein with Membranes

Considerable genetic and pathological evidence has implicated the small, soluble protein alpha-synuclein in the pathogenesis of familial and sporadic forms of Parkinsons disease (PD). However, the precise role of alpha-synuclein in the disease process as well as its normal function remain poorly understood. We recently found that an interaction with lipid rafts is crucial for the normal, pre-synaptic localization of alpha-synuclein. To understand how alpha-synuclein interacts with lipid rafts, we have now developed an in vitro binding assay to rafts purified from native membranes. Recapitulating the specificity observed in vivo, recombinant wild type but not PD-associated A30P mutant alpha-synuclein binds to lipid rafts isolated from cultured cells and purified synaptic vesicles. Proteolytic digestion of the rafts does not disrupt the binding of alpha-synuclein, indicating an interaction with lipid rather than protein components of these membranes. We have also found that alpha-synuclein binds directly to artificial membranes whose lipid composition mimics that of lipid rafts. The binding of alpha-synuclein to these raft-like liposomes requires acidic phospholipids, with a preference for phosphatidylserine (PS). Interestingly, a variety of synthetic PS with defined acyl chains do not support binding when used individually. Rather, the interaction with alpha-synuclein requires a combination of PS with oleic (18:1) and polyunsaturated (either 20:4 or 22:6) fatty acyl chains, suggesting a role for phase separation within the membrane. Furthermore, alpha-synuclein binds with higher affinity to artificial membranes with the PS head group on the polyunsaturated fatty acyl chain rather than on the oleoyl side chain, indicating a stringent combinatorial code for the interaction of alpha-synuclein with membranes.

The effects of ebselen on [ 3H]glutamate uptake by synaptic vesicles from rat brain

Ebselen is a selenium organic compound, which has been shown to be a neuroprotective agent in brain disorders involving glutamate receptors. However, the effects of ebselen on the functionality of the glutamatergic system are still poorly investigated. In this study, by using synaptic vesicle preparation, we investigated the effects of ebselen (0.3 to 10 μM) on (i) vesicular glutamate uptake, (ii) bafilomycin-sensitive H +-ATPase activity, and (iii) proton gradient formation (ΔpH). Ebselen presented a dual effect on glutamate uptake: the 1 μM concentration resulted in a 60% increase of the uptake, while the 10 μM concentration resulted in 60% inhibition. We also observed that ebselen (10 μM) inhibited the H +-ATPase activity and dissipated the ΔpH. The inhibitory effects of ebselen were prevented by dithiothreitol (DTT). These findings suggest that high concentrations of ebselen may oxidize the essential thiol groups of the H +-ATPase, which in turn affect its activity and compromise the vesicular glutamate uptake, and consequently lead to an impairment of the neural homeostasis.

Guanine derivatives modulate l-glutamate uptake into rat brain synaptic vesicles

Glutamate uptake into synaptic vesicles is driven by a proton electrochemical gradient generated by a vacuolar H +-ATPase and stimulated by physiological concentrations of chloride. This uptake plays an important role in glutamatergic transmission. We show here that vesicular glutamate uptake is selectively inhibited by guanine derivatives, in a time- and concentration-dependent manner. Guanosine, GMP, GDP, guanosine-5′- O-2-thiodiphosphate, GTP, or 5′-guanylylimidodiphosphate (GppNHp) inhibited glutamate uptake in 1.5 and 3 min incubations, however, when incubating for 10 min, only GTP or GppNHp displayed such inhibition. By increasing ATP concentrations, the inhibitory effect of GTP was no longer observed, but GppNHp still inhibited glutamate uptake. In the absence of ATP, vesicular ATPase can hydrolyze GTP in order to drive glutamate uptake. However, 5 mM GppNHp inhibited ATP hydrolysis by synaptic vesicle preparations. GTP or GppNHp decreased the proton electrochemical gradient, whereas the other guanine derivatives did not. Glutamate saturation curves were assayed in order to evaluate the specificity of inhibition of the vesicular glutamate carrier by the guanine derivatives. The maximum velocity of the initial rate of glutamate uptake was decreased by all guanine derivatives. These results indicate that, although GppNHp can inhibit ATPase activity, guanine derivatives are more likely to be acting through interaction with vesicular glutamate carrier.

Roles of ATP in depletion and replenishment of the releasable pool of synaptic vesicles.

Synaptic terminals of retinal bipolar neurons contain a pool of readily releasable synaptic vesicles that undergo rapid calcium-dependent release. ATP hydrolysis is required for the functional refilling of this vesicle pool. However, it was unclear which steps required ATP hydrolysis: delivery of vesicles to their anatomical release sites or preparation of synaptic vesicles and/or the secretory apparatus for fusion. To address this, we dialyzed single synaptic terminals with ATP or the poorly hydrolyzable analogue ATP-gammaS and examined the size of the releasable pool, refilling of the releasable pool, and the number of vesicles at anatomical active zones. After minutes of dialysis with ATP-gammaS, vesicles already in the releasable pool could still be discharged. This pool was not functionally refilled despite the fact that its anatomical correlate, the number of synaptic vesicles tethered to active zone synaptic ribbons, was completely normal. We conclude 1) because the existing releasable pool is stable during prolonged inhibition of ATP hydrolysis, whereas entry into the functional pool is blocked, a vesicle on entering the pool will tend to remain there until it fuses; 2) because the anatomical pool is unaffected by inhibition of ATP hydrolysis, failure to refill the functional pool is not caused by failure of vesicle movement; 3) local vesicle movements important for pool refilling and fusion are independent of conventional ATP-dependent motor proteins; and 4) ATP hydrolysis is required for the biochemical transition of vesicles and/or release sites to fusion-competent status.

Inhibition of Glutamate Uptake into Synaptic Vesicles from Rat Brain by 3-Nitropropionic Acid in Vitro

The exact mechanisms by which 3-nitropropionic acid (3-NP), a naturally occurring plant and fungal neurotoxin, exerts its neurotoxic effects are not fully understood. However, blockage of ATP synthesis by the irreversible inhibition of succinate dehydrogenase activity, increased production of free radicals, and secondary excitotoxicity have been implicated in its actions. In the present study, synaptic vesicle preparations from brain of adult rats were incubated with 3-NP at final concentrations ranging from 0.01 to 10 mM for the determination of glutamate uptake. The effect of 3-NP on γ-aminobutyric acid (GABA) and glycine uptake was also studied. Glutamate incorporation into vesicles was inhibited by 3-NP in a dose-dependent manner, whereas doses of up to 10 mM neurotoxin did not affect GABA or glycine uptake. Moreover, 3-NP did not inhibit the ATPase activity of synaptic vesicles. These findings indicate that low concentrations of 3-NP are able to selectively prevent vesicular glutamate storage, and this may represent at least one of the mechanisms responsible for the neurotoxic effects of 3-NP.

Inhibition of glutamate uptake into synaptic vesicles of rat brain by the metabolites accumulating in maple syrup urine disease

Maple syrup urine disease is an inherited metabolic disorder characterized by tissue accumulation of branched-chain amino acids and their corresponding keto acids in the affected children. Although this disorder is predominantly characterized by neurological symptoms, only few studies were carried out to investigate its neuropathology. In this study we investigated the effect of the metabolites accumulating in maple syrup urine disease on the in vitro uptake of [ 3H]glutamate by synaptic vesicles of rat brain. Synaptic vesicle preparations from whole brain of male adult Wistar rats (200–250 g) were incubated with the branched-chain amino acids and their corresponding keto acids at final concentrations ranging from 0.25 to 10 mM for the determination of glutamate uptake. Glutamate uptake was significantly inhibited by l-leucine, l-isoleucine, l-2-ketoisocaproic acid and l-2-keto-3-methylvaleric acid by approximately 60%, whereas l-valine and l-2-ketoisovaleric acid showed no effect. We also verified that the metabolites probably act by competitive inhibition. Therefore, it is possible that extracellular glutamate levels may be increased in maple syrup urine disease and that excitotoxicity may be involved in the neuropathology of this disorder.

Synaptobrevin 2 Is Palmitoylated in Synaptic Vesicles Prepared from Adult, But Not from Embryonic Brain

Neuronal SNARE-proteins such as synaptobrevin, SNAP 25, and synaptotagmin are key players during neurosecretion. So far palmitoylation of SNAP-25 and synaptotagmin 1 have been described in vivo. Here we have analyzed palmitoylation of the SNARE-proteins synaptobrevin 2 and synaptotagmin in vitro using synaptosomal and synaptic vesicle preparations from rat brain. Labeling of synaptic vesicles prepared from adult brain with [3H]palmitate revealed synaptobrevin 2 besides synaptotagmin 1 as major palmitoylated proteins. [3H]Palmitoylation of synaptobrevin 2 was resistant to chloroform/methanol extraction, but sensitive to reducing agents indicating a covalent fatty acid bond to cysteine residues. Palmitoylation of synaptobrevin 2 was also confirmed using endogenous synaptobrevin 2 present in PC-12 cells and synaptobrevin 2 expressed with a vacciniavirus system in Cos cells. In contrast to the situation seen with membrane preparations obtained from adult brain, synaptic vesicles prepared from embryonic rat brain did not support [3H]palmitoylation of synaptobrevin and synaptotagmin. These results suggest, that both synaptobrevin 2 and synaptotagmin were efficiently palmitoylated from mature synaptic vesicles. However, at least one component of the palmitoylation machinery is developmentally upregulated.

The Neuronal Monoamine Transporter VMAT2 Is Regulated by the Trimeric GTPase Go2

Monoamines such as noradrenaline and serotonin are stored in secretory vesicles and released by exocytosis. Two related monoamine transporters, VMAT1 and VMAT2, mediate vesicular transmitter uptake. Previously we have reported that in the rat pheochromocytoma cell line PC 12 VMAT1, localized to peptide-containing secretory granules, is controlled by the heterotrimeric G-protein Go(2). We now show that in BON cells, a human serotonergic neuroendocrine cell line derived from a pancreatic tumor expressing both transporters on large, dense-core vesicles, VMAT2 is even more sensitive to G-protein regulation than VMAT1. The activity of both transporters is only downregulated by Galphao(2), whereas comparable concentrations of Galphao(1) are without effect. In serotonergic raphe neurons in primary culture VMAT2 is also downregulated by pertussis toxin-sensitive Go(2). By electron microscopic analysis from prefrontal cortex we show that VMAT2 and Galphao(2) associate preferentially to locally recycling small synaptic vesicles in serotonergic terminals. In addition, Go(2)-dependent modulation of VMAT2 also works when using a crude synaptic vesicle preparation from this brain area. We conclude that regulation of monoamine uptake by the heterotrimeric G proteins is a general feature of monoaminergic neurons that controls the content of both large, dense-core and small synaptic vesicles.