Taar1-mediated modulation of presynaptic dopaminergic neurotransmission: role of D2 dopamine autoreceptors.

Trace Amine-Associated Receptor 1 (TAAR1) is a G protein-coupled receptor (GPCR) expressed in several mammalian brain areas and activated by "trace amines" (TAs). TAs role is unknown; however, discovery of their receptors provided an opportunity to investigate their functions. In vivo evidence has indicated an inhibitory influence of TAAR1 on dopamine (DA) neurotransmission, presumably via modulation of dopamine transporter (DAT) or interaction with the D2 DA receptor and/or activation of inwardly rectifying K(+) channels. To elucidate the mechanisms of TAAR1-dependent modulation, we used TAAR1 knockout mice (TAAR1-KO), a TAAR1 agonist (RO5166017) and a TAAR1 antagonist (EPPTB) in a set of neurochemical experiments. Analysis of the tissue content of TAAR1-KO revealed increased level of the DA metabolite homovanillic acid (HVA), and in vivo microdialysis showed increased extracellular DA in the nucleus accumbens (NAcc) of TAAR1-KO. In fast scan cyclic voltammetry (FSCV) experiments, the evoked DA release was higher in the TAAR1-KO NAcc. Furthermore, the agonist RO5166017 induced a decrease in the DA release in wild-type that could be prevented by the application of the TAAR1 antagonist EPPTB. No alterations in DA clearance, which are mediated by the DAT, were observed. To evaluate the interaction between TAAR1 and D2 autoreceptors, we tested the autoreceptor-mediated dynamics. Only in wild type mice, the TAAR1 agonist was able to potentiate quinpirole-induced inhibitory effect on DA release. Furthermore, the short-term plasticity of DA release following paired pulses was decreased in TAAR1-KO, indicating less autoinhibition of D2 autoreceptors. These observations suggest a close interaction between TAAR1 and the D2 autoreceptor regulation.

Monoamine transporter and receptor interaction profiles of a new series of designer cathinones.

Psychoactive ß-keto amphetamines (cathinones) are sold as "bath salts" or "legal highs" and recreationally abused. We characterized the pharmacology of a new series of cathinones, including methedrone, 4-methylethcathinone (4-MEC), 3-fluoromethcathinone (3-FMC), pentylone, ethcathinone, buphedrone, pentedrone, and N,N-dimethylcathinone. We investigated norepinephrine (NE), dopamine (DA), and serotonin (5-HT) uptake inhibition using human embryonic kidney 293 (HEK 293) cells that express the respective human monoamine transporter, the drug-induced efflux of NE, DA, and 5-HT from monoamine-preloaded cells, and binding affinity to monoamine transporters and receptors. All of the cathinones were potent NE uptake inhibitors but differed in their DA vs. 5-HT transporter inhibition profiles and monoamine release effects. Methedrone was a more potent 5-HT than DA transporter inhibitor and released NE and 5-HT similar to para-methoxymethamphetamine (PMMA), para-methoxyamphetamine (PMA), 4-methylthioamphetamine (4-MTA), and 3,4-methylenedioxymethamphetamine (MDMA). 4-MEC and pentylone equipotently inhibited all of the monoamine transporters and released 5-HT. Ethcathinone and 3-FMC inhibited NE and DA uptake and released NE, and 3-FMC also released DA similar to N-ethylamphetamine and methamphetamine. Pentedrone and N,N-dimethylcathinone were non-releasing NE and DA uptake inhibitors as previously shown for pyrovalerone cathinones. Buphedrone preferentially inhibited NE and DA uptake and also released NE. None of the cathinones bound to rodent trace amine-associated receptor 1, in contrast to the non-ß-keto-amphetamines. None of the cathinones exhibited relevant binding to other monoamine receptors. In summary, we found considerable differences in the monoamine transporter interaction profiles among different cathinones and compared with related amphetamines.

A new perspective for schizophrenia: TAAR1 agonists reveal antipsychotic- and antidepressant-like activity, improve cognition and control body weight.

Schizophrenia is a chronic, severe and highly complex mental illness. Current treatments manage the positive symptoms, yet have minimal effects on the negative and cognitive symptoms, two prominent features of the disease with critical impact on the long-term morbidity. In addition, antipsychotic treatments trigger serious side effects that precipitate treatment discontinuation. Here, we show that activation of the trace amine-associated receptor 1 (TAAR1), a modulator of monoaminergic neurotransmission, represents a novel therapeutic option. In rodents, activation of TAAR1 by two novel and pharmacologically distinct compounds, the full agonist RO5256390 and the partial agonist RO5263397, blocks psychostimulant-induced hyperactivity and produces a brain activation pattern reminiscent of the antipsychotic drug olanzapine, suggesting antipsychotic-like properties. TAAR1 agonists do not induce catalepsy or weight gain; RO5263397 even reduced haloperidol-induced catalepsy and prevented olanzapine from increasing body weight and fat accumulation. Finally, TAAR1 activation promotes vigilance in rats and shows pro-cognitive and antidepressant-like properties in rodent and primate models. These data suggest that TAAR1 agonists may provide a novel and differentiated treatment of schizophrenia as compared with current medication standards: TAAR1 agonists may improve not only the positive symptoms but also the negative symptoms and cognitive deficits, without causing adverse effects such as motor impairments or weight gain.

Pharmacological characterization of designer cathinones in vitro.

BACKGROUND AND PURPOSE: Designer ß-keto amphetamines (e.g. cathinones, 'bath salts' and 'research chemicals') have become popular recreational drugs, but their pharmacology is poorly characterized. EXPERIMENTAL APPROACH: We determined the potencies of cathinones to inhibit DA, NA and 5-HT transport into transporter-transfected HEK 293 cells, DA and 5-HT efflux from monoamine-preloaded cells, and monoamine receptor binding affinity. KEY RESULTS: Mephedrone, methylone, ethylone, butylone and naphyrone acted as non-selective monoamine uptake inhibitors, similar to cocaine. Mephedrone, methylone, ethylone and butylone also induced the release of 5-HT, similar to 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) and other entactogens. Cathinone, methcathinone and flephedrone, similar to amphetamine and methamphetamine, acted as preferential DA and NA uptake inhibitors and induced the release of DA. Pyrovalerone and 3,4-methylenedioxypyrovalerone (MDPV) were highly potent and selective DA and NA transporter inhibitors but unlike amphetamines did not evoke the release of monoamines. The non-ß-keto amphetamines are trace amine-associated receptor 1 ligands, whereas the cathinones are not. All the cathinones showed high blood-brain barrier permeability in an in vitro model; mephedrone and MDPV exhibited particularly high permeability. CONCLUSIONS AND IMPLICATIONS: Cathinones have considerable pharmacological differences that form the basis of their suggested classification into three groups. The predominant action of all cathinones on the DA transporter is probably associated with a considerable risk of addiction.

The norepinephrine transporter inhibitor reboxetine reduces stimulant effects of MDMA ("ecstasy") in humans.

This study assessed the pharmacodynamic and pharmacokinetic effects of the interaction between the selective norepinephrine (NE) transporter inhibitor reboxetine and 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") in 16 healthy subjects. The study used a double-blind, placebo-controlled crossover design. Reboxetine reduced the effects of MDMA including elevations in plasma levels of NE, increases in blood pressure and heart rate, subjective drug high, stimulation, and emotional excitation. These effects were evident despite an increase in the concentrations of MDMA and its active metabolite 3,4-methylenedioxyamphetamine (MDA) in plasma. The results demonstrate that transporter-mediated NE release has a critical role in the cardiovascular and stimulant-like effects of MDMA in humans.

Geldanamycin restores a defective heat shock response in vivo.

Induced expression of heat shock proteins (Hsps) plays a central role in promoting cellular survival after environmental and physiological stress. We have previously shown that scrapie-infected mouse neuroblastoma (ScN2a) cells fail to induce the expression of Hsp72 and Hsp28 after various stress conditions. Here we present evidence that this impaired stress response is due to an altered regulation of HSF1 activity. Upon stress in ScN2a cells, HSF1 was converted into hyperphosphorylated trimers but failed to acquire transactivation competence. A kinetic analysis of HSF1 activation revealed that in ScN2a cells trimer formation after stress was efficient, but disassembly of trimers proceeded much faster than in the uninfected cell line. Geldanamycin, a Hsp90-binding drug, significantly delayed disassembly of HSF1 trimers after a heat shock and restored stress-induced expression of Hsp72 in ScN2a cells. Heat-induced Hsp72 expression required geldanamycin to be present; following removal of the drug ScN2a cells again lost their ability to mount a stress response. Thus, our studies show that a defective stress response can be pharmacologically restored and suggest that the HSF1 deactivation pathway may play an important role in the regulation of Hsp expression.

Role played by sodium in activity-dependent secretion of neurotrophins - revisited.

In previous experiments, a causal relationship between sodium influx and secretion of nerve growth factor (NGF) was deduced from the observation that the sodium substitute N-methyl-D-glucamine (NMDG) abolished any activity-mediated NGF secretion that depends on intact internal calcium stores. However, all available experimental evidence speaks against sodium-mediated calcium mobilization from these stores under physiological conditions. We now report that rapid sodium influx initiated by monensin or ouabain did not induce brain-derived neurotrophic factor (BDNF) secretion from either native hippocampal slices or BDNF-transduced hippocampal neuronal cultures. Additionally, we found marked differences between the replacement of sodium by NMDG and sucrose on the one hand, and choline and lithium on the other. Replacement of 100% (and as little as 10%) sodium by NMDG or sucrose not only blocked the activity-mediated neurotrophin secretion, but itself led to a rapid and substantial increase of neurotrophin secretion. In contrast, the replacement of sodium (10% and 100%) by lithium and choline did not result in a release of neurotrophins, and only 100% replacement blocked the activity-mediated neurotrophin secretion. We conclude that the blocking effects of NMDG and sucrose on neurotrophin secretion do not reflect the sodium replacement, but instead represent an independent blocking effect. These differences were also reflected in part by electrophysiological investigations in individually patched hippocampal neurons. The importance of the present observations lies not only in the reevaluation of the involvement of sodium in activity-dependent neurotrophin secretion, but also in the demonstration that sodium replacement may initiate 'side effects' that are unrelated to sodium replacement.

Are there differences between the secretion characteristics of NGF and BDNF? Implications for the modulatory role of neurotrophins in activity-dependent neuronal plasticity.

In previous experiments the activity-dependent secretion of nerve growth factor (NGF) from native hippocampal slices and from NGF-cDNA transfected hippocampal neurons showed unusual characteristics [Blochl and Thoenen (1995) Eur J Neurosci 7:1220-1228; (1996) Mol Cell Neurosci 7:173-190]. In both hippocampal slices and cultured hippocampal neurons the activity-dependent secretion proved to be independent of extracellular calcium, but dependent on the release of calcium from intracellular stores. Under different experimental conditions, Goodman et al. [(1996) Mol Cell Neurosci 7:222-238] reported that the high potassium-mediated secretion of brain-derived neurotrophic factor (BDNF) from hippocampal cultures was dependent on extracellular calcium. Mowla et al. [(1997) Proc 27th Annu Meet Soc Neurosci New Orleans 875.10] reported on even further-reaching differences between NGF and BDNF secretion, namely, that in hippocampal neurons and in pituitary cell lines NGF was secreted exclusively according to the constitutive pathway, whereas BDNF was exclusively sorted according to the activity-dependent regulated pathway. In view of the crucial importance of such potential differences between the processing, sorting, and secretory mechanisms of different neurotrophins for their modulatory roles in activity-dependent neuronal plasticity, a thorough analysis under comparable experimental conditions was mandatory. We demonstrate that in native hippocampal slices and adenoviral-transduced hippocampal neurons there are no differences between NGF and BDNF with respect to the subcellular distribution and mechanism of secretion; that the activity-dependent secretion of both NGF and BDNF is dependent on intact intracellular calcium stores; and that the differences between our own observations and those of Goodman et al. (ibid.) regarding the dependence on extracellular calcium do not reflect differences between NGF and BDNF sorting and secretion, but reflect the differing experimental conditions used.

Partial cortical devascularization results in elevations of cortical nerve growth factor and increases nerve growth factor protein within basal forebrain cholinergic neurons.

Previous studies have demonstrated that partial cortical devascularization results in increased levels of nerve growth factor protein within the tissue immediately surrounding the infarcted region. In the present study, we have used this lesion model to further characterize the nerve growth factor increase by investigating: (i) the time course for this phenomenon; (ii) the impact of the devascularizing lesion on cortical regions not directly impinged upon by the lesion; and (iii) the response of nerve growth factor-sensitive nucleus basalis neurons providing afferent cortical innervation to the increased availability of nerve growth factor within their target territory. Our results indicate that, within the infarcted cortex, nerve growth factor levels increase rapidly following the lesion (up 51% by one day post lesion), reach a maximum of 136% above controls by three days and undergo a slow decline back to baseline levels by 23 days. Within the frontal and cingulate cortices, which are not devascularized by the lesion and show no signs of pathological damage, nerve growth factor levels increase over a similar time course, and with a similar magnitude, to those in the lesioned cortex. Nerve growth factor-sensitive nucleus basalis neurons on the side ipsilateral to the lesion respond to increased cortical nerve growth factor levels with an increased accumulation of nerve growth factor within their cell bodies (revealed by nerve growth factor immunohistochemistry and quantitative enzyme-linked immunosorbent assay) which was apparent at three days following the lesion, but no longer discernible at seven or 14 days or later. The present study investigated a model of cortical devascularization for its ability to alter nerve growth factor levels within the cortex. Nerve growth factor levels were rapidly increased within the infarcted cortical tissue beneath the lesion but were also elevated to a similar extent, and with a similar time course, in cortical regions not directly impinged upon by the lesion. The retrograde impact of elevated cortical nerve growth factor levels was demonstrated by an increased accumulation of nerve growth factor within the cell bodies of nucleus basalis neurons providing innervation to the cortex. This lesion model should provide a potential avenue for investigating the functional role(s) of nerve growth factor in the intact and lesioned adult central nervous system, as well as the internal mechanisms for regulating its synthesis, release, uptake, and degradation.

Reversible sedimentation and masking of nerve growth factor (NGF) antigen by high molecular weight fractions from rat brain.

Nerve growth factor (NGF) was recently found to be largely associated with sedimentable fractions of adult rat brain and treatments of the fractions by alkaline pH increased the measurable amount of their NGF antigen as well as its solubilization [M.C. Hoener, E. Hewitt, J.M. Conner, J.W. Costello and S. Varon, Nerve growth factor (NGF) content in adult rat brain tissues is several-fold higher than generally reported and is largely associated with sedimentable fractions, Brain Res., 728 (1996) 47-56; M.C. Hoener and S. Varon, Effects of sodium chloride, Triton X-100, and alkaline pH on the measurable contents and sedimentability of the nerve growth factor (NGF) antigen in adult rat hippocampal tissue extracts, J. Neurosci. Res., in press (1997); C. Zettler, D.C.McL. Bridges, X.-F. Zhou and R.A. Rush, Detection of increased tissue concentrations of nerve growth factor with improved extraction procedure, J. Neurosci. Res., 46 (1996) 581-594]. We have further investigated the reversibility of these pH effects. Reversal of the pH of an adult rat hippocampal tissue extract from 10.5 to 7.4 led to an almost complete transfer of NGF back from nonsedimentable to sedimentable fractions and to a remasking of the previously unmasked portion of NGF antigen. Thus, molecules causing masking and sedimentation of NGF at pH 7.4 were likely to be present in the alkaline extract. A gel filtration column in PBS, pH 10.5 was used to separate such putative binding molecules from the NGF. All of the NGF antigen from rat hippocampal alkaline extract was found to elute with 19 kDa fractions. The same apparent molecular weight was found for mouse submaxillary beta-NGF and recombinant human beta-NGF. Masking and sedimentation no longer occurred when newly generated 19 kDa rat brain NGF was returned to pH 7.4. When high molecular weight fractions derived from the same gel filtration (in PBS, pH 10.5) were added back to the 19 kDa NGF pool at pH 7.4 and the mixture incubated and centrifuged, the measurability of 19 kDa rat brain NGF antigen was markedly reduced and half of the antigen was recovered in sedimentable fractions. Similar but less dramatic results were obtained when mixing the same high molecular weight fractions with 19 kDa mouse or human beta-NGF. These findings provide new opportunities to identify molecules to which NGF may be bound within intact brain tissues.

Effects of sodium chloride, Triton X-100, and alkaline pH on the measurable contents and sedimentability of the nerve growth factor (NGF) antigen in adult rat hippocampal tissue extracts.

Nerve growth factor (NGF) was found to be largely associated with sedimentable fractions of hippocampal and other neural tissues of the adult rat (Hoener et al.: Brain Res 728:47-56, 1996), verified by both bioassay and ELISA techniques. In the present study, the ELISA assay conditions were improved and simplified. Bovine serum albumin was needed in the phosphate buffered saline for maximal measurability of NGF antigen. Hippocampal tissue sonicates were separated into nonsedimentable supernatant and sedimentable pellet fractions. Individual or combined treatments with sodium chloride, Triton X-100, and pH were applied to the samples for possible effects on the i) measurable content of NGF antigen and ii) distribution of sedimentable and nonsedimentable forms. The amount of measurable NGF antigen was found to be increased in a dose dependent fashion by sodium chloride between 0.15 and 0.35 M, Triton X-100 between 0 and 0.5%, and pH between 8.5 and 10.5. The same treatment that led to maximal measurable NGF levels (0.7% Triton X-100 and pH 10.5) also caused the release of the NGF antigen from sedimentable to nonsedimentable fractions. Similar findings regarding maximal NGF antigen levels and release were seen for treatments applied to the sonicate before separation into a supernatant and pellet fraction.

Nerve growth factor (NGF) content in adult rat brain tissues is several-fold higher than generally reported and is largely associated with sedimentable fractions.

Initial studies had revealed that the bioactivity of nerve growth factor (NGF) in sonicates of adult rat hippocampal formation (HF) is several-fold greater in their pellet than their supernatant fractions. Such observations have prompted an analysis of NGF antigen (NGF-Ag) contents in pellets and supernatants from a variety of adult rat CNS tissues, both in the absence and the presence of exogenous beta-NGF. With HF tissues, NGF-Ag in the supernatants was comparable to most literature values, but pellet NGF-Ag was 3 to 5 times that amount. All other CNS tissue sonicates also revealed 3-6 fold higher NGF-Ag in their pellets than their supernatants, hence overall NGF-Ag contents were greatly in excess of reported ones. Presentation of mouse beta-NGF to a tissue, its sonicate, or its standard pellet resulted in a transfer to the final pellet of 30-50% of the added soluble NGF-Ag (and 30% of the added bioactivity). This percentage is much lower than that present in native pellets (80%), suggesting that the association of endogenous NGF with particulate matter may involve at least two compartments. Treatments of pellets with salt, alkaline pH, and/or the detergent Triton X-100 have revealed a third subset, namely additional pellet NGF-Ag that was not initially recognized by the antibody in our ELISA assay. The treatments also caused substantial release of NGF from pellet to supernatant. Further studies should clarify the nature of the association between NGF and the three subsets of pellet NGF and allow the investigation of the pellet molecules responsible for it.

Glycosyl-phosphatidylinositol-specific phospholipase D. Interaction with and stimulation by apolipoprotein A-I.

Glycosyl-phosphatidylinositol-specific phospholipase D (GPI-PLD) is an amphiphilic protein which, in serum, is associated with high-density lipoproteins (HDL). It is shown that the major component of the HDL fraction, apolipoprotein A-I (apo A-I), is responsible for this association. In the absence of apo A-I, purified GPI-PLD occurred as virtually inactive aggregates which became disaggregated by apo A-I. The enzyme/apo A-I complex efficiently hydrolyzed the solubilized GPI-anchored substrate, acetylcholinesterase. Triton X-100 was also able to dissociate aggregated GPI-PLD, however, it strongly inhibited enzyme activity at detergent concentrations above the critical micellar concentration.

Phosphatidylinositol-glycan-specific phospholipase D is an amphiphilic glycoprotein that in serum is associated with high-density lipoproteins.

Phosphatidylinositol (PtdIns)-glycan-specific phospholipase D was purified from bovine and human serum by phase separation in Triton X-114 and by chromatography on DEAE-cellulose, octyl-Sepharose, concanavalin-A-Sepharose, and hydroxyapatite. The purification of the two enzymes was approximately 1200-fold with a recovery of 3-5%. Bovine serum contained about 40 micrograms/ml of PtdIns-glycan-specific phospholipase D, about 10 times more than the amount determined in human serum. PtdIns-glycan-specific phospholipase D is also present in mammalian cerebrospinal fluid and in mammalian milk but to a much lesser extent than in serum. Enzyme from bovine and human serum displayed amphiphilic properties as revealed by sucrose density gradient centrifugation and gel filtration in the absence and presence of detergent. On density gradient centrifugation, both enzymes sedimented with an apparent sedimentation coefficient of about 6.0 S in the presence of 0.1% Triton X-100, and formed aggregates up to 14.5 S in the absence of detergent. Upon gel filtration, the bovine and human enzymes migrated with a Stokes' radius of 6.5 nm and 6.6 nm, respectively, in the presence of Triton X-100. In the absence of Triton X-100, both enzymes gave a Stokes' radius of 8.8 nm. Serial centrifugation of serum at increasing NaBr concentrations revealed that the majority of the enzyme is contained in the high-density lipoprotein fraction. PtdIns-glycan-specific phospholipase D from bovine and human serum contained 27 and 28 N-acetylglucosamine residues, respectively. Treatment with N-glycosidase F decreased the apparent molecular mass of the bovine and human enzyme from 115 and 123 kDa to 91 and 87 kDa, respectively. Sequence analysis of peptides derived from PtdIns-glycan-specific phospholipase D of bovine serum by CNBr cleavage gave 100% identity to the sequence published for the bovine liver enzyme while there was 83% similarity and 74% identity to the sequence of peptides obtained from the human serum enzyme.

Isolation and characterization of a phosphatidylinositol-glycan-anchor-specific phospholipase D from bovine brain.

In recent years an increasing number of proteins has been shown to be membrane-anchored by a covalently attached PtdIns-glycan residue. In mammalian cells little is known about PtdIns-glycan-specific phospholipases which might play a role in the metabolism of PtdIns-glycan-anchored proteins. In order to identify PtdIns-glycan-specific phospholipases, a rapid and sensitive assay for such enzymes was developed using the PtdIns-glycan-anchored amphiphilic membrane form of acetylcholinesterase as substrate. The rate of product formation was monitored by the increase in soluble hydrophilic acetylcholinesterase in the aqueous phase after separation in Triton X-114. With this assay we established the presence of a PtdIns-glycan-specific phospholipase in bovine brain. This enzyme was soluble and could be partially purified by a heat step followed by chromatography on DEAE-cellulose and by gel filtration on Sepharose CL-6B. PtdIns-glycan-specific phospholipase had a high affinity for the PtdIns-glycan anchor of the substrate (Km = 52 nM) and did not degrade either PtdCho or PtdIns. Hydrophobic labeling of the anchor of the substrate with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) caused a marked decrease in the cleavage rate and methylation of the amino group of the glucosamine residue of the anchor decreased the cleavage rate to zero. Using [125I]TID-labeled substrate, diradylglycerol phosphate was identified as the second product showing that the cleavage specificity of PtdIns-glycan-specific phospholipase was that of a phospholipase D. PtdIns-glycan-specific phospholipase D was inhibited by mercurials, omicron-phenanthroline and EGTA. It was stimulated by Ca2+ in micromolar concentrations indicating that PtdIns-glycan-phospholipase D is a Ca2(+)-regulated enzyme.