Journal of Neurochemistry

Published by Wiley
Online ISSN: 1471-4159
Publications
Article
Ammonium trichloro(dioxoethylene-0,0') tellurate (AS101) is a non-toxic organotellurium compound with pleiotropic activities. It was recently shown to induce production of the neurotrophic factor glial cell line-derived neurotrophic factor and to rescue neuronal-like PC-12 cells from neurotrophic factor deprivation-induced apoptosis. In this study, we show that AS101 improves functional outcome and reduces brain damage in a mouse model of focal ischemic stroke. Both pre-stroke and post-stroke intraperitoneal treatments with AS101 reduced infarct size and edema and improved the neurological function of the animals. AS101 treatments reduced both apoptotic and inflammatory caspase activities, and also inhibited protein tyrosine nitration suggesting that AS101 suppresses oxidative stress. Studies of cultured neurons showed that AS101 confers protection against apoptosis induced by either glucose deprivation or the lipid peroxidation product 4-hydroxynonenal. Moreover, AS101 treatment reduced glutamate-induced intracellular calcium elevation, a major contributor to neuronal death in stroke. As AS101 has an excellent safety profile in humans, our pre-clinical data suggest a potential therapeutic benefit of AS101 in patients suffering from stroke and other neurodegenerative conditions.
 
Article
Although members of the multiple vertebrate/mammalian dopamine D1 receptor gene family can be selectively classified on the basis of their molecular/phylogenetic, structural, and tissue distribution profiles, no subtype-specific discriminating agents have yet been identified that can functionally differentiate these receptors. To define distinct pharmacological/functional attributes of multiple D1-like receptors, we analyzed the ligand binding profiles, affinity, and functional activity of 12 novel NNC compounds at mammalian/vertebrate D1/D1A and D5/D1B, as well as vertebrate D1C/D1D, dopamine receptors transiently expressed in COS-7 cells. Of all the compounds tested, only NNC 01-0012 displayed preferential selectivity for vertebrate D1C receptors, inhibiting [3H]SCH-23390 binding with an estimated affinity (approximately 0.6 nM) 20-fold higher than either mammalian/vertebrate D1/D1A or D5/D1B receptors or the D1D receptor. Functionally, NNC 01-0012 is a potent antagonist at D1C receptors, inhibiting to basal levels dopamine (10 microM)-stimulated adenylyl cyclase activity. In contrast, NNC 01-0012 (10 microM) exhibits weak antagonist activity at D1A receptors, inhibiting only 60% of maximal cyclic AMP production by dopamine, while acting as a partial agonist at vertebrate D1B and D1D receptors, stimulating adenylyl cyclase activity by approximately 33% relative to the full agonist dopamine (10 microM), an effect that was blocked by the selective D1 receptor antagonist NNC 22-0010. These data clearly suggest that the benzazepine NNC 01-0012, despite lacking the N-methyl residue in the R3 position, is a selective and potent D1C receptor antagonist. Moreover, the differential signal transduction properties exhibited by NNC 01-0012 at these receptor subtypes provide further evidence, at least in vertebrates, for the classification of the D1C receptor as a distinct D1 receptor subtype.
 
Article
In this study we evaluated UCN-01, a small molecule that inhibits protein kinases by interacting with the ATP-binding site, as a potential anti-cancer agent for neuroblastoma. UCN-01 was effective at inducing apoptosis in six neuroblastoma cell lines with diverse cellular and genetic phenotypes. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assays, detection of active caspase-3 and cleaved poly ADP-ribose polymerase (PARP) confirmed that UCN-01 induced apoptosis. Cell cycle analysis determined that the UCN-01 treated cells accumulated in S phase by 16 h. Unlike vinblastine and docetaxel that increased survivin expression, UCN-01 treatment did not increase X-linked inhibitor of apoptosis protein (XIAP) and survivin levels. Analysis of specific phosphoepitopes on chk1/2, Akt, and GSK3beta following UCN-01 treatment determined that there was no significant change in phospho-chk1/2. However, there was decreased immunoreactivity at Ser473 and Thr308 of Akt and Ser9 of GSK3beta by 4 h indicating that the Akt survival pathway and downstream signalling was compromised. Thus, UCN-01 was effective at inducing apoptosis in neuroblastoma cell lines.
 
Article
Using primary cultures of bovine adrenal chromaffin cells labelled with 32Pi, we show that stimulation with bradykinin, nicotine, or a depolarising concentration of potassium stimulates the accumulation of [32P]phosphatidic acid. The effects of nicotine and potassium are smaller than the effect of bradykinin, and are dependent entirely on extracellular calcium. The diacylglycerol kinase inhibitor R 59 022 attenuates the formation of phosphatidic acid by nicotine and depolarising concentrations of potassium. This inhibitor also blocks the nicotine and potassium stimulation of noradrenaline release from chromaffin cells. Using 45Ca2+ influx studies, we show that the nicotine-evoked calcium influx is also attenuated by R 59 022. These observations contrast with those in another report in which we showed that bradykinin stimulation of either [32P]phosphatidic acid accumulation or noradrenaline release is not affected by R 59 022. It is likely that the calcium influx produced by nicotine and depolarising potassium is blocked by R 59 022 by a mechanism that is independent of its ability to block diacylglycerol kinase. The nicotine- and potassium-stimulated [32P]phosphatidic acid accumulation is a consequence of this calcium influx and presumably reflects calcium activation of either phospholipase C or phospholipase D.
 
Article
Feedback inhibition of tyrosine hydroxylase by catechols was evaluated using in situ and in vitro enzyme assays. The three catechol compounds used were norepinephrine, 2-hydroxyestradiol, and 3'4'-dihydroxy-2-methylpropiophenone (U-0521, Upjohn); representing endogenous catecholamines, catechol estrogens, and a synthetic catechol, respectively. The in situ experiments were performed with dissociated retinal cells from rats and with stationary phase adrenergic-like neuroblastoma cells (N1E-115). The catechol estrogen, 2-hydroxyestradiol, resembled the endogenous catecholamine in its potency to inhibit in vitro and in situ tyrosine hydroxylations with IC50 values of 10 microM in vitro and 100 microM in situ. The drug U-0521, which has been used as an inhibitor of catechol-O-methyltransferase (COMT), was also found to be an inhibitor of tyrosine hydroxylase. Further, it was shown to be more potent than the natural catechols, both in vitro and in situ, with IC50 values of 30--600 nM.
 
Article
The effects of the selective dopamine D2 receptor antagonists YM-09151-2 and l-sulpiride on the in vivo release of dopamine (DA), L-3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in rat striatum were investigated. The drugs were injected into the striatum through a microinjection needle attached to a dialysis probe. YM-09151-2 (0.1 or 1.0 microgram/0.5 microliter) injected into the striatum produced a dramatic rapid-onset transient increase in striatal DA release in a dose-dependent manner. However, the DA increase induced by l-sulpiride (15 or 75 ng/0.5 microliter) was small and of slower onset. An increase of DOPAC levels by YM-09151-2 was biphasic: The first peak occurred at 40 min, followed by a delayed-onset gradual increase. Slower-onset gradual increases were also found in DOPAC levels after l-sulpiride injection and in HVA levels after injections of both YM-09151-2 and l-sulpiride. The infusion of tetrodotoxin (TTX; 2 microM) revealed two different types of DA release mechanisms: The rapid-onset transient DA release induced by YM-09151-2 was TTX insensitive, whereas the slower-onset DA release induced by l-sulpiride was TTX sensitive. Moreover, the rapid-onset transient DA release was Ca2+ independent and was not affected by pretreatment with l-sulpiride or nomifensine. Therefore, it is concluded that YM-09151-2 injected into the striatum produced a transient striatal DA release that is independent of D2 receptors and the action potential.
 
Article
The addition of nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) to PC12 cells prelabeled with [3H]inositol and preincubated for 15 min in the presence of 10 mM LiCl stimulated the production of inositol phosphates with maximal increases of 120-180% in inositol monophosphate (IP), 130-200% in inositol bisphosphate (IP2), and 45-50% in inositol trisphosphate (IP3) within 30 min. The majority of the overall increase (approximately 85%) was in IP; the remainder was recovered as IP2 and IP3 (approximately 10% as IP2 and 5% as IP3). Under similar conditions, carbachol (0.5 mM) stimulated about a 10-fold increase in IP, a sixfold increase in IP2, and a fourfold increase in IP3. The mass level of 1,2-diacylglycerol (DG) in PC12 cells was found to be dependent on the incubation conditions; in growth medium [Dulbecco's modified Eagle's medium (DME) plus serum], it was around 6.2 mol %, in DME without serum, 2.5 mol %, and after a 15-min incubation in Dulbecco's phosphate-buffered saline, 0.62 mol %. The addition of NGF and bFGF induced an increase in the mass level of DG of about twofold within 1-2 min, often rising to two- to threefold by 15 min, and then decreasing slightly by 30 min. This increase was dependent on the presence of extracellular Ca2+, and was inhibited by both phenylarsine oxide (25 microM) and 5'-deoxy-5'-methylthioadenosine (3 mM). Under similar conditions, 0.5 mM carbachol stimulated the production of DG to the same extent as 200 ng/ml NGF and 50 ng/ml bFGF. Because carbachol is much more effective in stimulating the production of inositol phosphates, the results suggest that both NGF and bFGF stimulate the production of DG primarily from phospholipids other than the phosphoinositides.
 
Article
The uptake of [14C]ethylenediamine into slices of rat brain and its subsequent evoked release have been studied. An active uptake process was demonstrated by comparing uptake at 37 and 4 degrees C. This uptake showed a Km of 1.36 mM, was partly sodium-dependent and was reduced by nipecotic acid. Release could be readily evoked by 30 mM potassium, and by electrical stimulation, the release in both cases being calcium-dependent. In view of these findings and the reported interactions of ethylenediamine with gamma-aminobutyric acid-related mechanisms, it might be of interest to determine whether this simple diamine occurs endogenously in the mammalian brain.
 
Article
The relative suitability of different molecular species of 1,2-diacyl-sn-glycerols as substrates for the diacylglycerol kinase (ATP: 1,2-diacyl-sn-glycerol phosphotransferase) in rat brain microsomes was investigated. The diacylglycerols tested were a mixture of the 1-[3H]palmitoyl and 1-[14C]stearoyl homologues of either the 2-oleoyl (monoenoic), 2-linoleoyl (dienoic), 2-arachidonoyl (tetraenoic), or 2-docosahexaenoyl (hexaenoic) diacylglycerols with an isotope ratio (3H/14C) approximately equal to 1.00. At substrate concentrations of 0.125 mM and 0.60 mM, only a modest preference of the kinase for total (1-palmitoyl plus 1-stearoyl homologues) monoenoic over total hexaenoic species was indicated. The tetraenoic diacylglycerols gave reaction rates which were not significantly different from the monoenes, dienes, or hexaenes when the data were analyzed statistically. No significant enzyme selectivity for either the 1-palmitoyl or 1-stearoyl homologues of the various 1-saturated 2-unsaturated diacylglycerols was apparent. The present results, together with data on the composition of free 1,2-diacylglycerols in brain, which reveal a preponderance of tetraenoic molecular species, suggest that the tetraenoic phosphatidic acids (mainly as 1-stearoyl 2-arachidonoyl species) are quite possibly the major products of diacylglycerol kinase activity in rat brain under physiological conditions.
 
Article
The axonal outgrowth of cells of Neuro2a, a mouse neuroblastoma cell line, was suppressed on expression of the beta-galactoside alpha 1,2-fucosyltransferase (alpha 1,2-FT) gene. We recently cloned two types of rabbit alpha 1,2-FT, RFT-I and RFT-II. RFT-I exhibits comparable kinetic properties and structural homology with human H gene alpha 1,2-FT, and RFT-II shows comparable kinetic parameters with human Se gene alpha 1,2-FT. Neuro2a cells expressing RFT-I (N2A-RFT-I) contained a large amount of fucosyl GM1 instead of GM1 and GD1a, major gangliosides in the parent Neuro2a cells, whereas Neuro2a cells expressing RFT-II (N2A-RFT-II) showed a subtle change in the ganglioside pattern. N2A-RFT-II and parent Neuro2a cells showed axonal outgrowth in serum-free medium on the exogenous addition of GM1, whereas N2A-RFT-I cells exhibited multiple neurite sprouts but not axonal outgrowth. This phenotype was fully recovered by N2A-RFT-I cells on the addition of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol and alpha-L-fucosidase to the culture medium, which resulted in pronounced reduction of fucosyl GM1 expression. These results suggested that expression of H-type alpha1,2-FT, and subsequent incorporation of fucose into glycolipids and glycoproteins, especially the formation of fucosyl GM1, modifies the response of neuronal cells to stimuli that induce axonal extension.
 
Article
Highly purified rat brain myelin isolated by two different procedures showed appreciable activity for CDP-ethanolamine: 1,2-diacyl-sn-glycerol ethanolaminephosphotransferase (EC 2.7.8.1). Specific activity was close to that of total homogenate and approximately 12-16% that of brain microsomes. Three other lipid-synthesizing enzymes, cerebroside sulfotransferase, lactosylceramide sialyltransferase, and serine phospholipid exchange enzyme, were found to have less than 0.5% the specific activity in myelin compared with microsomes. Washing the myelin with buffered salt or taurocholate did not remove the phosphotransferase, but activity was lost from both myelin and microsomes by treatment with Triton X-100. It resembled the microsomal enzyme in having a pH optimum of 8.5 and a requirement for Mn2+ and detergent, but differed in showing no enhancement with EGTA. The diolein Km was similar for the two membranes (2.5-4 x 10(-4) M), but the CDP-ethanolamine Km was lower for myelin (3-4 x 10(-5) M) than for microsomes (11 - 13 x 10(-5 M). Evidence is reviewed that this enzyme is able to utilize substrate from the axon in situ.
 
Article
We have applied the 19F-nuclear magnetic resonance (NMR) calcium indicator 1,2-bis(2-amino-5-fluoro-phenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA) to the measurement of the free intracellular calcium concentration [( Ca2+]i) in superfused brain slices. A mean +/- SD control value of 380 +/- 71 nM (n = 18) was obtained at 37 degrees C using 2.4 mM extracellular Ca2+. Subcellular fractionation studies using [3H]5FBAPTA showed that after loading of its tetraacetoxymethyl ester, approximately 55% was de-esterified, with the other 45% remaining as the tetraester bound to membranes. Of the de-esterified 5FBAPTA, greater than 90% was in the cytosolic fractions, with less than 1% in the mitochondria or microsomes. The NMR-visible de-esterified 5FBAPTA slowly disappeared from the tissue with a t1/2 of 4 h. A time course after loading confirmed that the calculated [Ca2+]i was constant over a 5-h period, although the scatter of individual results was +/- 20%. The [Ca2+]i was increased by a high extracellular K+ concentration ([K+]e), by a low extracellular concentration of Na+, and by the calcium ionophore A23187. On recovery from high [K+]e, the [Ca2+]i "overshot" to values lower than the original control value. The [Ca2+]i was surpisingly resistant to changes in extracellular Ca2+ concentration.
 
The Srxn1 promoter contains an ARE which is induced by Nrf2 expression in cortical neurons, as is the endogenous gene. (a) Schematic showing the putative ARE within mammalian Srxn1 promoters. (b) Effect of Nrf2 expression on wild-type and mutant Srxn1-Luc reporters (normalized to Renilla control-see methods). Neurons were transfected with Srxn1 reporter plus pcDNA3.1-Nrf2 or pGlobin control plasmid. NB. In this and all experiments ‘con’ denotes pGlobin control vector.*p < 0.05 (2-tailed paired t-test, n = 3–5). (c) Effect of Nrf2 expression on ARE-Luc activity. *p < 0.05 (n = 5). (d) Effect of transfecting pEF-Nrf2 on endogenous Srxn1 expression. Neurons were transfected with the indicated vectors and after 24 h subjected to immunocytochemical analysis of sulfiredoxin expression. Immunofluorescence performed as described (McKenzie et al. 2006). Scale bar = 40 μm. (e) Effect of Nrf2 expression on WT and mutant Srxn1-Luc reporters in glial cells *p < 0.05 (n = 7). (f) Effect of Nrf2 expression on ARE-Luc activity in glial cells. *p < 0.05 (n = 4). (g) Effect of Nrf2 expression on WT and mutant Srxn1-Luc reporters in HEK293cells. *p < 0.05 (n = 3).
Basal levels of Srxn1 promoter activity are sensitive to Keap1 expression in glia but not neurons. (a,b) Effect of Keap1 expression on basal and Nrf2-driven ARE-Luc reporter activity in neurons (a) and glia (b). Cells were transfected with ARE-Luc plus pcDNA3.1-Nrf2 (or pGlobin), plus pcDNA3.1-Keap1 (or pGlobin). *p < 0.05 (n = 4–6). (c,d) Effect of Keap1 expression on basal Srxn1-Luc reporter activity in neurons (c) and glia (d). *p < 0.05 (n = 4).
Activity-dependent induction of Srxn1 via the ARE/AP-1 site is primarily mediated by AP-1. (a) Effect of Keap1 expression on activity-dependent induction of reporters of Srxn1 and Sesn2. Neurons were transfected with the reporter plus TK-driven Renilla control vector. Twenty four hours post-transfection, bursts of action potential firing were induced by treatment of neurons with 50 μM bicuculline, and burst frequency was enhanced by addition of 250 μM 4-amino pyridine (Hardingham et al. 2001). Reporter activity was assayed after the indicated time periods. *p < 0.05 Bonferonni two-tailed paired t-test (compared to control, BiC/4-AP-stimulated level, n = 6). (b) Effect of an interfering mutant of AP-1 (TAM67) on activity-dependent induction of the putative ARE/AP-1 composite site, compared to an upstream AP-1 site. Neurons were transfected with the indicated reporter constructs, plus pTK-RL and either control vector or expression vector for TAM67. Twenty four hours post-transfection neurons were stimulated with BiC/4-AP overnight prior to assay of reporter activity (n = 5).
Article
Peroxiredoxins are an important family of cysteine-based antioxidant enzymes that exert a neuroprotective effect in several models of neurodegeneration. However, under oxidative stress they are vulnerable to inactivation through hyperoxidation of their active site cysteine residues. We show that in cortical neurons, the chemopreventive inducer 3H-1,2-dithiole-3-thione (D3T), that activates the transcription factor Nuclear factor erythroid 2-related factor (Nrf2), inhibits the formation of inactivated, hyperoxidized peroxiredoxins following oxidative trauma, and protects neurons against oxidative stress. In both neurons and glia, Nrf2 expression and treatment with chemopreventive Nrf2 activators, including D3T and sulforaphane, up-regulates sulfiredoxin, an enzyme responsible for reducing hyperoxidized peroxiredoxins. Induction of sulfiredoxin expression is mediated by Nrf2, acting via a cis-acting antioxidant response element (ARE) in its promoter. The ARE element in Srxn1 contains an embedded activator protein-1 (AP-1) site which directs induction of Srxn1 by synaptic activity. Thus, raising Nrf2 activity in neurons prevents peroxiredoxin hyperoxidation and induces a new member of the ARE-gene family, whose enzymatic function of reducing hyperoxidized peroxiredoxins may contribute to the neuroprotective effects of Nrf2 activators.
 
Article
The alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) is a peptide-coupling agent that is being used to inactivate irreversibly alpha 2-adrenoceptors and other receptors. The aim of the present study was to assess the in vitro and in vivo effects of EEDQ on the newly discovered brain I2-imidazoline sites, located mainly in mitochondria. Preincubation of rat cortical membranes with EEDQ (10(-8)-10(-5) M) markedly decreased (20-90%) the specific binding of the selective antagonist [3H]RX821002 to alpha 2-adrenoceptors without affecting that of [3H]idazoxan (in the presence of adrenaline) to I2-imidazoline sites. In EEDQ-pretreated membranes (10(-5) M, 30 min at 25 degrees C), the density of I2-imidazoline sites (Bmax = 80 +/- 4 fmol/mg of protein) was not different from that determined in untreated membranes in the presence of (10(-6) M (-)-adrenaline (Bmax = 83 +/- 4 fmol/mg of protein), and both densities were lower (24%, p < 0.05) than the total native density of [3H]idazoxan binding sites (Bmax = 107 +/- 6 fmol/mg of protein) (I2-imidazoline sites plus alpha 2-adrenoceptors). Treatment of rats with an optimal dose of EEDQ (1.6 mg/kg, i.p., for 2 h to 30 days) reduced maximally at 6 h (by 95 +/- 1%) the specific binding of [3H]-RX821002 to alpha 2-adrenoceptors, but also the binding of [3H]idazoxan to I2-imidazoline sites (by 44 +/- 5%). Pretreatment with yohimbine (10 mg/kg, i.p.) fully protected against EEDQ-induced alpha 2-adrenoceptor inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)
 
Article
The content of 1,2-diacylglycerol (DAG) was determined in sciatic nerves from normal and streptozotocin-induced diabetic rats. In nerves frozen in situ, DAG content was reduced 22% in the proximal region and 77% in the distal region of diabetic nerve, principally because of the loss of associated fat. DAG levels in freshly dissected and desheathed diabetic nerve were decreased from 23 to 30% as compared with normal nerve. Determination of DAG molecular species distribution in desheathed normal nerve indicated that 18:0/20:4 accounted for 34%, 16:0/18:1 for 17%, and several other polyunsaturated fatty acid-containing species for 17% of the total. In diabetic nerve, the quantity of the 18:0/20:4 DAG, species was reduced by 37%, and this drop was 62% of the reduction in all molecular species. The content of the minor species, 16:0/20:4 DAG, was decreased by 48%. Our results suggest that nerve DAG arises in large part from phosphoinositide degradation. Moreover, these results provide support for the hypothesis that reduced Na+,K(+)-ATPase activity in diabetic nerve is a consequence of decreased phosphoinositide turnover, which thereby generates insufficient DAG to maintain a protein kinase C-mediated step necessary for activation of Na+,K(+)-ATPase.
 
Article
N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) treatment, both in vitro and in vivo, results in an irreversible blockade of cortical S2 5-hydroxytryptamine (serotonin) receptors. Incubation of rat cortical homogenates with EEDQ in vitro results in a concentration-dependent (EC50 approximately 5 microM) and time-dependent decrease in the Bmax of [3H]ketanserin-labeled S2 serotonin receptors. Extensive washing of the homogenate following in vitro or in vivo EEDQ treatment does not result in an increase in the amount of [3H]ketanserin binding, indicating that EEDQ acts to modify irreversibly cortical S2 serotonin receptors. That the modification of S2 receptor binding by EEDQ occurs at the recognition site of the receptor is indicated by the finding that coincubation with the S2 receptor antagonist ketanserin, but not the D2 3,4-dihydroxyphenylethylamine (dopamine) receptor antagonist domperidone, selectively protects against the irreversible blockade of S2 serotonin receptors. Peripheral administration of EEDQ results in a dose-dependent reduction in cortical S2 serotonin receptors with maximal effects (approximately 90% reduction) observed following 10 mg/kg (i.p.). Seven days following peripheral administration of EEDQ there is a recovery of S2 serotonin receptors back to 74% of the original receptor population. These data demonstrate that EEDQ in vitro and in vivo acts as an irreversible antagonist of S2 serotonin receptors and that it can be used to investigate the recovery rate of these receptors.
 
Article
1,2-Diacyl-sn-glycerol (DAG) is a product of cell activation that has emerged as an important intracellular messenger whose primary function appears to be the activation of protein kinase C. They originate by the activation of phospholipases, which hydrolyze different phospholipids depending on the external stimulus and the nature of the cells, leading to the production of different molecular species. In the present study the quantitative changes in the total mass and the molecular species of DAG formed on phorbol ester (12-O-tetradecanoyl-phorbol 13-acetate) stimulation were investigated in proliferating and retinoic acid (RA)-differentiated human LA-N-1 cells. The TPA treatment of both cell types elicited an increase in the total amount of DAG. The increase was biphasic; i.e., an initial peak at 2-5 min was followed by a sustained increase that persisted for > 30 min. The analysis of the molecular species of DAG and phospholipids showed that in proliferating LA-N-1 cells, the DAG increase corresponds to the production of mainly saturated/monounsaturated (16:0-18:1, 18:0-18:1) and saturated/saturated (16:0-16:0, 16:0-18:0) species, suggesting that they originate essentially from the hydrolysis of phosphatidylcholine. In contrast, RA-differentiated cells responded to TPA treatment by increasing the level of saturated/polyunsaturated (16:1-22:6, 18:0-22:6, 16:0-20:4, 18:0-20:4) and monounsaturated/monounsaturated (18:1-18:1) species, suggesting mainly a phosphatidylethanolamine origin. These findings indicate that the treatment of LA-N-1 cells with TPA generates different molecular species of DAG depending on their physiological state. These observations suggest in turn that different phospholipases are activated by TPA in proliferating and RA-differentiated cells.
 
Article
Specific radioactivities of molecular species of phosphatidyl choline(PC), phosphatidyl ethanolamine(PE) and 1,2-diacylglycerol were determined in rabbit brain 15 and 30 min after intraventricular injection of 10OpCi of either [U-14C]glucose or [U-14C]glycerol. The rate of de nouo synthesis of glycerophospholipids and their molecular species could be determined after glycerol labelling, since 94.0–99.7% of 14C activity was recovered in glyceryl moieties of brain lipids. After injection of glucose radioactivity was measured in both glyccrol and acyl residues of lipids. High incorporation rates were measured in species of PC, PE and 1,2-diacylglycerol with oleic acid in position 2 and with palmitic, stearic or oleic acids in position 1. The conclusion may therefore be drawn that these molecular species were preferably synthesized de novo by selective acylation of glycerol 3-phosphate. The lowest specific activities were observed for 1,2-dipalmitoyl- and l-stearoyl-2- arachidonoyl-glycerol, -PC and -PE. These turnover rates point to incorporation of arachidonate, and probably also of palmitate in dipalmitoyl-PC, amounting to 20% of total PC, via deacylation-acylation- cycle.
 
Article
The neurons of dorsal root ganglia (DRG) mediate several sensation modalities. The carbohydrate antigens on DRG neurons differ with the sensation modalities that subsets of neurons convey. Despite the important roles of gangliosides and glycoproteins in neuronal differentiation and neuritogenesis of the mammalian nervous system, little is known about the mechanisms underlying the regulation of glycosylation. We previously demonstrated the expression of H-blood type antigens (Fuc alpha1, 2Gal beta) on rabbit DRG neurons of small diameter and dramatic changes in H antigens during the perinatal period. To investigate the possible biological roles and regulatory mechanisms of H antigens, we recently cloned three types of rabbit alpha1,2-fucosyltransferase gene that catalyze the biosynthesis of H antigens. Here, we analyze the expression of these genes, RFT-I, II, and III, in rabbit DRG. The H-type alpha1,2-fucosyltransferase gene, RFT-I, was expressed in DRG in late embryos to adult rabbits, as detected on northern blotting. The other two secretor-type alpha1,2-fucosyltransferase genes, RFT-II and III, were observed to be expressed in late embryonic DRG on RT-PCR analysis but were not detectable on northern blotting. The expression of the H-type alpha1,2-fucosyltransferase gene was analyzed by in situ hybridization and was found to be abundant in small-diameter DRG neurons. These results indicate that the H-type alpha1,2-fucosyltransferase gene plays a major role in the regulation of the H antigen expression in DRG during the perinatal period.
 
Article
The irreversible protein-modifying reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to investigate binding site characteristics on the gamma-aminobutyric acidA (GABAA) receptor complex. In vitro, preincubation with EEDQ led to a concentration-dependent decrease in receptor number for benzodiazepine, t-butylbicyclophosphorothionate (TBPS), and GABA binding sites in cerebral cortex. The effect was maximal at the highest concentration of EEDQ used (10(-4) M) and was greatest for the benzodiazepine site. Pretreatment of membranes with the benzodiazepine antagonist Ro 15-1788, 1 or 10 microM, or the agonist lorazepam, 10 microM, largely prevented the effects of EEDQ. Scatchard analysis indicated no effect of EEDQ, 10(-4) M, on apparent affinity, but a decrease in receptor density for each site. Administration of EEDQ to mice, 12.5 mg/kg i.p., led to a substantial (55-65%) decrease in number of benzodiazepine binding sites in cortex after 4 h. Slightly smaller changes were observed for TBPS and GABA binding. No changes were observed in apparent affinity at any site. Prior administration of Ro 15-1788, 5 mg/kg, prevented the effect of EEDQ on benzodiazepine binding. Density of benzodiazepine binding sites gradually recovered over time, and receptor density returned to control values by 96 h after EEDQ injection. Number of binding sites in cortex for TBPS and GABA also increased over time after EEDQ. Benzodiazepine sites in cerebellum were decreased proportionally to cortex after EEDQ, and increased over a similar time course. Function of the GABAA receptor in chloride uptake in cortex was markedly reduced (65%) by EEDQ.(ABSTRACT TRUNCATED AT 250 WORDS)
 
Article
1,2-Diacyl-, 1-alk-1'-enyl-2-acyl- and 1-alkyl-2-acyl-sn-glycero-3- phosphorylcholine specifically labelled with different acids at the 2 positions, were prepared enzymically using the acyltransferase system of rabbit sarcoplasmic reticulum. The substrates were submitted to hydrolysis by phospholipase A2 (phospholipid acyl-hydrolase, EC 3.1.1.4) obtained from normal and brain tissue affected with subacute sclerosing panencephalitis. In the diseased tissue an increase of phospholipase A2 activity ranging from 46 to 54% could be observed in comparison to the control brain for all substrates investigated. Among the investigated substrates, phospholipase A2 had the highest affinity for the 1,2 diacylcompound, whereas alkenylacyl- and alkylacyl-sn-glycero-3-phosphorylcholine were cleaved at almost similar rates. The hydrolysis rate of choline plasmalogen and the corresponding diacyl compound by the enzyme was greatly influenced by the fatty acid moiety located at the 2 position of the substrates.
 
Article
Gerbil forebrains were frozen in situ to inactivate the tissues, and 1,2-diacylglycerols were first measured quantitatively by HPLC. Although 1,2-diacylglycerols were completely recovered from the HPLC column, the control amount of 1,2-diacylglycerol in gerbil forebrain was only 79.6 nmol/g wet weight, which is about one-fourth of that previously reported for gerbil brain inactivated by liquid N2 after decapitation instead of in situ freezing. The fatty acid composition of 1,2-diacylglycerols in gerbil forebrain was first reported and the control 1,2-diacylglycerols were richer in palmitic acid than in stearic acid or arachidonic acid, which is rather different from the data previously reported for mouse or rat brain obtained by decapitation and analyzed by traditional TLC methods. The amount of 1,2-diacylglycerol increased by 82.9% in gerbil forebrain during 5 min of ischemia induced by bilateral carotid ligation. Arachidonic acid and stearic acid were abundant in the 1,2-diacylglycerols produced by 5 min of ischemia. Thus we were able to obtain accurate values of the amount and the fatty acid composition of 1,2-diacylglycerols in gerbil forebrains using HPLC and in situ freezing technique.
 
Article
The object of the present study was to determine if a reduced amount of lipid in the Quaking mouse brain were accompanied by decreased activities of any or all of the enzymes in the de novo synthesis of phosphatidylcholine via the CDP-choline pathway. In contrast to expectations, the results show a significant (P<0.005) increase in the cytosolic activity of the cholinephosphate cytidylyltransferase. In addition, this is the first report of these enzyme activities in mouse brain.
 
Article
J. Neurochem. (2012) 121, 115–124. We developed 1-(2-[18F]fluoro-3-pyridyl)-4-(2-isopropyl-1-oxo-isoindoline-5-yl)-5-methyl-1H-1,2,3-triazole ([18F]FPIT) as a promising positron emission tomography (PET) ligand for in vitro and in vivo imaging of metabotropic glutamate receptor type 1 (mGluR1) in rat and monkey brains. In vitro autoradiography with [18F]FPIT was used to determine the distribution of radioactivity in rat and monkey brains. In vivo experiments were performed using dissection and small-animal PET on rats, and PET on monkey. Metabolite analysis was performed on rat plasma and brain, and monkey plasma. Autoradiography of rat and monkey brains showed that [18F]FPIT binding is aligned with the reported distribution of mGluR1 with high specific binding in the cerebellum and thalamus. PET study on rat and monkey showed high brain uptake and distribution patterns consistent with those seen in the autoradiographic studies. The radioactivity in the brain was significantly decreased by pre-treatment with unlabeled FPIT, indicative of a specific signal for mGluR1 that was inhibited by mGluR1-selective ligand JNJ-16259865 in the brain. Metabolite analysis showed that the percentage of unchanged [18F]FPIT was 89% in the brain homogenate of rat at 90 min after injection. In the monkey plasma, the percentage of unchanged form was 50% at 90 min. [18F]FPIT produced in vitro and in vivo signals to visualize mGluR1 expression in rat and monkey brains, suggesting the usefulness of [18F]FPIT for imaging mGluR1 in human brain.
 
Article
In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mouse brain, there was no significant increase or decrease in the content of an endogenous amine, 1,2,3,4-tetrahydroisoquinoline (TIQ), which is well noted for inducing parkinsonism, whereas another endogenous amine, 1-methyl-1,2,3,4-tetrahydroisoquinoline (1-MeTIQ), was markedly reduced. This result agrees with the finding in human idiopathic parkinsonianism, confirmed by our previous research. In addition, pretreatment with 1-MeTIQ completely prevented MPTP- or TIQ-inducing bradykinesia, a symptom of parkinsonism. This study confirmed that 1-MeTIQ plays an important role in preventing the pathogenesis of parkinsonism and is a possible leading compound of anti-parkinsonism agents.
 
Article
Substances that mimic the actions of causative gene products of familial Parkinson's disease (PD) are candidate as causative agents of idiopathic PD. 1-Benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ), an endogenous neurotoxin, is present at three times higher levels in CSF of PD patients than in CSF of control subjects. However, the mechanism of 1BnTIQ's neurotoxicity is unclear. In this study, we tried to identify 1BnTIQ-binding proteins by using a diazido-functionalized 1BnTIQ analog, 1-(3-azido-5-azidomethylbenzyl)-1,2,3,4-tetrahydroisoquinoline, designed and synthesized as a probe for radioisotope-free photoaffinity labeling. One major photolabeled protein identified using this probe was tubulin beta, which has been reported to be a substrate of parkin, a ubiquitin E3 ligase and a causative gene product of familial PD. Loss of function mutation of parkin is reported to result in loss of tubulin beta ubiquitination. Therefore, we examined the effect of 1BnTIQ on ubiquitination of tubulin beta. The polyubiquitinated tubulin beta level in human neuroblastoma SH-SY5Y cells was reduced in the presence of 1BnTIQ, even at concentrations as low as those detected in parkinsonian CSF. In vitro ubiquitination assay gave similar results. It is suggested that 1BnTIQ has the same effect on tubulin ubiquitination as does mutant parkin in familial PD. Taken together, substances which reduce polyubiquitination of tubulin such as 1BnTIQ are supposed to be candidates of etiological factors of PD.
 
Article
Repeated amphetamine administration to rats under chronic ethanol intoxication resulted in the formation of 1,3-dimethyl-1,2,3,4-tetrahydroisoquinoline (1,3-DiMeTIQ), a novel metabolite of amphetamines. 1,3-DiMeTIQ was quantified with a sensitive, specific assay using gas chromatography-mass spectrometry. It was not found in the brains of rats given repeated amphetamine administration but no ethanol. The chronic ethanol-intoxicated rats subjected to repeated amphetamine administration exhibited behavioral abnormalities, such as repeated convulsions and curving of the back. 1,3-DiMeTIQ contents were markedly higher in the brain or plasma of rats manifesting abnormal behavior in comparison with those in rats behaving normally. Thus, the 1,3-DiMeTIQ content in the rat brain seems to have some relationship with behavioral abnormalities. This study also confirmed that 1,3-DiMeTIQ can cross the blood-brain barrier in the rat. Intraperitoneal 1,3-DiMeTIQ injections to rats caused behavioral symptoms similar to those observed in rats with chronic ethanol intoxication and repeated amphetamine administration. The effect of toxic doses of 1,3-DiMeTIQ on dopaminergic and serotonergic metabolism in the whole rat brain was also investigated.
 
Article
N-Methyl-1,2,3,4-tetrahydroisoquinoline (NMTIQ) was found to be oxidized by monoamine oxidase (MAO) into N-methylisoquinolinium ion, which was proved to inhibit enzymes related to the metabolism of catecholamines, such as tyrosine hydroxylase, aromatic-L-amino acid decarboxylase, and MAO. NMTIQ was oxidized by both types A and B MAO in human brain synaptosomal mitochondria. Oxidation was dependent on the amount of MAO sample and the reaction time. Enzyme activity with respect to NMTIQ reached optimum at a pH of approximately 7.25, as was the case with other substrates. Type A MAO had higher activity for this substrate than type B. The Km and Vmax values of the oxidation by types A and B MAO were 571 +/- 25 microM and 0.29 +/- 0.06 pmol/min/mg protein, and 463 +/- 43 microM and 0.16 +/- 0.03 pmol/min/mg protein, respectively. The Vmax values of types A and B MAO for NMTIQ were much smaller than those for other substrates such as kynuramine. NMTIQ was the first tetrahydroisoquinoline shown to be oxidized into the isoquinolinium ion by MAO in the brain.
 
Article
1-Methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ), unlike several other tetrahydroisoquinolines, displays neuroprotective properties. To elucidate this action we compared the effects of 1MeTIQ with 1,2,3,4-tetrahydroisoquinoline (TIQ), a compound sharing many activities with 1MeTIQ (among them reducing free radicals formed during dopamine catabolism), but offering no clear neuroprotection. We found that the compounds similarly inhibit free-radical generation in an abiotic system, as well as indices of neurotoxicity (caspase-3 activity and lactate dehydrogenase release) induced by glutamate in mouse embryonic primary cell cultures (a preparation resistant to NMDA toxicity). However, in granular cell cultures obtained from 7-day-old rats, 1MeTIQ prevented the glutamate-induced cell death and 45Ca2+ influx, whereas TIQ did not. This suggested a specific action of 1MeTIQ on NMDA receptors, which was confirmed by the inhibition of [3H]MK-801 binding by 1MeTIQ. Finally, we demonstrated in an in vivo microdialysis experiment that 1MeTIQ prevents kainate-induced release of excitatory amino acids from the rat frontal cortex. Our results indicate that 1MeTIQ, in contrast to TIQ, offers a unique and complex mechanism of neuroprotection in which antagonism to the glutamatergic system may play a very important role. The results suggest the potential of 1MeTIQ as a therapeutic agent in various neurodegenarative illnesses of the central nervous system.
 
Article
1-Benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ) was detected as a novel endogenous amine in mouse brain and parkinsonian CSF by using the gas chromatography-selected ion-monitoring method. The level of 1BnTIQ was very high in CSF of some parkinsonian patients compared with that of controls with other neurological diseases, the mean value being three times higher (parkinsonians: 1.17 +/- 0.35 ng/ml of CSF, n = 18; vs. controls: 0.40 +/- 0.10 ng/ml of CSF, n = 11; mean +/- SEM, not significantly different). The pole test, a toxicological examination to evaluate behavior abnormalities related to Parkinson's disease, was used to examine the pharmacological effect of 1BnTIQ in mice. Repeated administration of 1BnTIQ induced behavior abnormalities, which pretreatment with 1-methyl-1,2,3,4-tetrahydroisoquinoline could prevent. We suggest that 1BnTIQ may be related to the idiopathic Parkinson's disease.
 
Article
Trichloroethylene, a common industrial solvent and a metabolic precursor of chloral hydrate, occurs widely in the environment. Chloral hydrate, which is also used as a hypnotic, has been found to condense spontaneously with tryptamine, in vivo, to give rise to a highly unpolar 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) that has a structural analogy to the dopaminergic neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Earlier studies have revealed the relative permeability of the molecule through the blood-brain barrier and its ability to induce Parkinson-like symptoms in rats. In this study, we report that TaClo induces an apoptotic pathway in the human neuroblastoma cell line, SK-N-SH, involving the translocation of mitochondrial cytochrome c to the cytosol and activation of caspase 3. TaClo-induced apoptosis shows considerable differences from that mediated by other Parkinson-inducing agents such as MPTP, rotenone and manganese. Although it is not clear if the clinically administered dosage of chloral hydrate or the relatively high environmental levels of trichloroethylene could lead to an onset of Parkinson's disease, the spontaneous in vivo formation of TaClo and its pro-apoptotic properties, as shown in this report, should be considered.
 
Article
The effect of single and multiple 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) and 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ) administration on concentrations of dopamine and its metabolites: homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxytyramine (3MT) in three brain areas was studied HPLC with electrochemical detection in Wistar rats. The rate of dopamine catabolism in the striatum along the N-oxidative and O-methylation pathways was assessed by calculation of the ratio of appropriate metabolites to dopamine concentration. In addition, the spontaneous and apomorphine-stimulated locomotor activity, and muscle rigidity was studied after acute administration of 1MeTIQ and 1BnTIQ. We have found that 1MeTIQ did not change the level of dopamine and HVA in all investigated structures both after a single and chronic administration. However, the levels of intermediary dopamine metabolites, DOPAC and 3MT, were distinctly affected. The level of DOPAC was strongly depressed (by 60-70%) while the level of extraneuronal matabolite 3MT was significantly elevated (by 170-200%). In contrast to 1MeTIQ, 1BnTIQ depressed the level of dopamine (by approximately 60%) and increased the level of total metabolite, HVA, (by 40%) especially in the striatum, but the levels of DOPAC and 3MT remained unchanged. The paper has shown that 1MeTIQ and 1BnTIQ produced different effects on dopamine catabolism. Potential neuroprotective compound 1MeTIQ did not change the rate of total dopamine catabolism, it strongly inhibited the monoamine oxidase (MAO)-dependent catabolic pathway and significantly activated the catechol-O-methyltransferase (COMT)-dependent O-methylation. In contrast 1BnTIQ, a compound with potential neurotoxic activity, produced the significant increase of the rate of dopamine metabolism with strong activation of the oxidative MAO-dependent catabolic pathway. Interestingly, both compounds produced similar antidopaminergic functional effects: antagonism of apomorphine hyperactivity and induction of muscle rigidity. The results may explain the biochemical basis of the neuroprotective and of the neurotoxic properties endogenous brain tetrahydroisoquinoline derivatives.
 
Article
On the basis of affinity differences for spiperone, two binding sites for [3H](+/-)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene ([3H]ADTN) in the rat brain could be distinguished: "D3" with a low and "D4" with a high affinity for spiperone. Evidence is provided that D3 and D4 sites are related to high agonist affinity states of the D1 and D2 dopamine receptors, respectively. Various well-known selective D1 and D2 agonists and antagonists showed potencies at these sites in agreement with this hypothesis. A comparison of the Bmax values for [3H]ADTN binding to D3 and D4 sites with the numbers of D1 receptors (labelled by [3H]SCH 23390) and of D2 receptors (labelled by [3H]spiperone), both in the striatum and in the mesolimbic system, indicated that under the conditions used for 3H-agonist binding experiments, both populations of D1 and D2 receptors were converted to their high agonist affinity states to a considerable, although different extent. In fact, when competition experiments with [3H]spiperone were performed under the conditions otherwise used for [3H]ADTN binding experiments (instead of the conditions usually used for antagonist binding), substantial shifts of the displacement curves of 3,4-dihydroxyphenylethylamine (dopamine) and ADTN toward higher affinities were observed. A comparison of the effects of various agonists and antagonists in the [3H]ADTN binding experiments and in functional tests revealed a significant correlation between their potencies at D4 binding sites and at D2 receptors modulating the release of [3H]acetylcholine from striatal slices. However, in the situation of the D1/D3 pair, when the measurement of adenylate cyclase activity was taken as a functional test for D1 receptors, agonists were more active in the binding than in the functional test, whereas for many antagonists the opposite was found. The results are discussed with regard to the classification and functional aspects of brain dopamine receptors.
 
Article
Salsolinol is one of the dopamine-derived tetrahydroisoquinolines and is synthesized from pyruvate or acetaldehyde and dopamine. As it cannot cross the blood-brain barrier, salsolinol as the R enantiomer in the brain is considered to be synthesized in situ in dopaminergic neurons. Effects of R and S enantiomers of salsolinol on kinetic properties of tyrosine hydroxylase [tyrosine, tetrahydrobiopterin:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2], the rate-limiting enzyme of catecholamine biosynthesis, were examined. The naturally occurring cofactor of tyrosine hydroxylase, L-erythro-5,6,7,8-tetrahydrobiopterin, was found to induce allostery to the enzyme polymers and to change the affinity to the biopterin itself. Using L-erythro-5,6,7,8-tetrahydrobiopterin, tyrosine hydroxylase recognized the stereochemical structures of the salsolinols differently. The asymmetric center of salsolinol at C-1 played an important role in changing the affinity to L-tyrosine. The allostery of tyrosine hydroxylase toward biopterin cofactors disappeared, and at low concentrations of biopterin such as in brain tissue, the affinity to the cofactor changed markedly. A new type of inhibition of tyrosine hydroxylase, by depleting the allosteric effect of the endogenous biopterin, was found. It is suggested that under physiological conditions, such a conformational change may alter the regulation of DOPA biosynthesis in the brain.
 
Article
We designed as candidate metabolites and synthesized two 1-benzyl-1,2,3,4-tetrahydroisoquinoline derivatives containing a dopamine moiety: 1-(3',4'-dihydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline (3',4'DHBnTIQ) and 1-benzyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (6,7DHBnTIQ). Both were detected in mouse brain as endogenous amines by gas chromatography/mass spectrometry. 3',4'DHBnTIQ induced parkinsonism in mice when chronically administered intraperitoneally, whereas 6,7DHBnTIQ did not despite the structural similarity of the two compounds. This difference may be related to cellular uptake: In rat striatal synaptosomes, these compounds were intracellularly transported by the dopamine transporter with Km values of 6.14 and 7.82 microM and Vmax values of 214.3 and 112.2 pmol/min/mg of protein, respectively. Thus, endogenous 3',4'DHBnTIQ may be actively transported into dopaminergic neurons and accumulated there, contributing at least in part to the induction of idiopathic Parkinson's disease.
 
Article
N-Methylation of (R)-1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [(R)-salsolinol] derived from dopamine was proved by in vivo microdialysis study in the rat brain. The striatum was perfused with (R)-salsolinol and N-methylated compound was identified in the dialysate using HPLC and electrochemical detection with multichanneled electrodes. N-Methylation of (R)-salsolinol was confirmed in three other regions of the brain, the substantia nigra, hypothalamus, and hippocampus. In the substantia nigra, the amount of N-methylated (R)-salsolinol was significantly larger than in the other three regions. These results indicate that around dopaminergic neurons, particularly in the substantia nigra, (R)-salsolinol was methylated into N-methyl-(R)-salsolinol, which has a chemical structure similar to that of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, the selective dopaminergic neurotoxin. N-Methylation of tetrahydroisoquinolines and beta-carbolines have already been proven to increase their toxicity to dopaminergic neurons and N-methylation might be an essential step for these alkaloids to increase their toxicity. On the other hand, after perfusion of (R)-salsolinol, release of dopamine and 5-hydroxytryptamine was observed and inhibition of monoamine oxidase was indicated. (R)-Salsolinol and its derivatives may be candidates for being dopaminergic neurotoxins.
 
Article
The endogenous monoamine 1-methyl-1,2,3,4-tetrahydroisoquinoline has been shown to prevent the neurotoxic effect of MPP(+) and other endogenous neurotoxins, which produce a parkinsonian-like syndrome in humans. We have tested its potential protective effect in vivo by measuring the protection of 1-methyl-1,2,3,4-tetrahydroisoquinoline in the neurotoxicity elicited by MPP(+) in rat striatum by tyrosine hydroxylase immunocytochemistry. Because we know that cellular damage caused by MPP(+) is primarily the result of mitochondrial respiratory inhibition at the complex I level, we have extended the study further to understand this protective mechanism. We found that the inhibitory effect on the mitochondrial respiration rate induced by MPP(+) in isolated rat liver mitochondria and striatal synaptosomes was prevented by addition of 1-methyl-1,2,3,4-tetrahydroisoquinoline. This compound has no antioxidant capacity; therefore, this property is not involved in its protective effect. Thus, we postulate that the preventive effect that 1-methyl-1,2,3,4-tetrahydroisoquinoline has on mitochondrial inhibition for MPP(+) could be due to a "shielding effect," protecting the energetic machinery, thus preventing energetic failure. These results suggest that this endogenous amine may protect against the effect of several parkinsonism-inducing compounds that are associated with progressive impairment of the mitochondrial function.
 
Article
The deposition of amyloid beta (Abeta) protein is a consistent pathological hallmark of Alzheimer's disease (AD) brains; therefore, inhibition of Abeta fibril formation and destabilization of pre-formed Abeta fibrils is an attractive therapeutic and preventive strategy in the development of disease-modifying drugs for AD. This study demonstrated that Paeonia suffruticosa, a traditional medicinal herb, not only inhibited fibril formation of both Abeta(1-40) and Abeta(1-42) but it also destabilized pre-formed Abeta fibrils in a concentration-dependent manner. Memory function was examined using the passive-avoidance task followed by measurement of Abeta burden in the brains of Tg2576 transgenic mice. The herb improved long-term memory impairment in the transgenic mice and inhibited the accumulation of Abeta in the brain. Three-dimensional HPLC analysis revealed that a water extract of the herb contained several different chemical compounds including 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranose (PGG). No obvious adverse/toxic were found following treatment with PGG. As was observed with Paeonia suffruticosa, PGG alone inhibited Abeta fibril formation and destabilized pre-formed Abeta fibrils in vitro and in vivo. Our results suggest that both Paeonia suffruticosa and its active constituent PGG have strong inhibitory effects on formation of Abeta fibrils in vitro and in vivo. PGG is likely to be a safe and promising lead compound in the development of disease-modifying drugs to prevent and/or cure AD.
 
Article
Mouse brain synaptosomes, essentially devoid of mitochondrial contamination, were used as a model to study the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) on the levels of ATP of neuronal terminals. Similar to known inhibitors of ATP synthesis, both MPTP and MPP+ caused a dramatic depletion of synaptosomal ATP. This depletion was dose dependent and occurred as a relatively early biochemical event in the absence of any apparent damage to synaptosomal membranes. MPP+ was more effective than its parent compound in decreasing ATP; it induced a significant loss at concentrations (10-100 microM) similar to those it reaches in the brain in vivo. MPTP-induced ATP depletion was completely prevented by the monoamine oxidase B inhibitor deprenyl, which, on the contrary, was ineffective against MPP+. As expected in view of the heterogeneous population of nerve terminals present in our synaptosomal preparations, the catecholamine uptake blocker mazindol did not significantly affect the ATP loss caused by both compounds. Data indicate that (1) administration of MPTP may cause a depletion of ATP within neuronal terminals resulting from the generation of MPP+, and (2) exposure to the levels of MPP+ reached in vivo may cause biochemical changes that are nonselective for dopaminergic terminals.
 
Article
Twenty analogs of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were tested for their capacity to be oxidized by pure monoamine oxidase-A (MAO-A) prepared from human placenta and pure monoamine oxidase-B (MAO-B) prepared from beef liver. Several of the MPTP analogs were very good substrates for MAO-A, for MAO-B, or for both and had low Km values and high turnover numbers. These values were similar to or even better than those of kynuramine and benzylamine, good substrates for MAO-A and MAO-B, respectively. MPTP had relatively low Km values for oxidation by both MAO-A and MAO-B. In contrast, the turnover number for MPTP oxidation by MAO-B was considerably higher than the value for MAO-A. The corresponding pyridinium species of MPTP and several of the MPTP analogs inhibited MAO-A competitively with Ki values at micromolar concentrations; in contrast the pyridinium species inhibited MAO-B competitively at considerably higher concentrations (i.e., 100 microM or greater Ki values). The data provide information concerning the structural requirements for the oxidation of tetrahydropyridines by MAO-A and MAO-B and the inhibition of these enzymes by pyridiniums.
 
Article
The effect of the neurotoxin 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) was investigated in mouse N2a neuroblastoma cells, induced to differentiate by serum withdrawal and addition of dibutyryl cyclic AMP, over a 24-h period. Addition of MPTP (10 microM) during differentiation caused a change in cell morphology characterised by an inhibition of axon outgrowth, in the absence of cell death. Biochemical characterisation by western blotting revealed that MPTP had no significant effects on the levels of actin, alpha-tubulin, or total heavy-chain neurofilament (NF-H). However, NF-H phosphorylation appeared to increase following MPTP treatment when blots were probed with the phosphorylation state-specific antibodies RMd09 and Ta51. In addition, indirect immunofluorescence analysis revealed an accumulation of phosphorylated NF-H in the cell perikaryon, suggesting that altered NF-H distribution was associated with the observed effects of MPTP on cell morphology. These changes may represent a useful in vitro marker of MPTP neurotoxicity within a simple differentiating neuronal cell model system.
 
Article
1-Methyl-4-benzyl-1,2,3,6-tetrahydropyridine (MBzTP), an analogue of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, despite its rapid oxidation by monoamine oxidase B (MAO B), is not neurotoxic. The pyridinium expected to arise from the four-electron oxidation of MBzTP inhibits mitochondrial respiration and the oxidation of NADH in inner membranes and is only moderately less inhibitory than 1-methyl-4-phenylpyridinium. It is also a competitive inhibitor of dopamine uptake by the dopamine transporter and hence likely to be taken up into neurons, despite its relatively high Ki value (Ki = 21 microM). Incubation of MBzTP with purified MAO B yields first the dihydropyridinium form, then a mixture of the pyridinium form and another unidentified product, in proportions that depend on the concentrations of MAO B and oxygen. At low MAO B concentration and moderate oxygen concentration, nonenzymatic formation of the unidentified product predominates. The lack of neurotoxicity of MBzTP appears to be due to the oxidative destruction of the dihydropyridine and consequent failure of accumulation of 1-methyl-4-benzylpyridinium.
 
Article
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 30 mg/kg i.p. daily for 7 days, was administered to mice. This dosage regimen resulted in an approximately 50% reduction of striatal dopamine (DA) level. Chronic administration of GM1 ganglioside (II3NeuAc-GgOse Cer), beginning between 1 to 4 days after terminating MPTP dosing, resulted in partial restoration of the striatal DA level. From dose- and time-response studies, it appeared that 30 mg/kg i.p. of GM1 administered daily for approximately 23 days resulted in an approximately 80% restoration of the DA level and complete restoration of the 3,4-dihydroxyphenylacetic acid (DOPAC) content. This dosage of GM1 also restored the turnover rate of DA in the striatum to near normal. Discontinuing GM1 treatment resulted in a fall of DA and DOPAC levels to values found in mice treated with MPTP alone. There was no evidence for regeneration of nerve terminal amine reuptake in the GM1-treated mice as evaluated by DA uptake into synaptosomes. Our biochemical findings in animals suggest that early GM1 ganglioside treatment of individuals with degenerative diseases of dopaminergic nigrostriatal neurons might be fruitful.
 
Article
Treatment of common marmosets with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 1-4 mg/kg for up to 4 days) caused a profound parkinsonian state. Ten days from the start of MPTP treatment, all animals showed marked motor impairment, consisting of bradykinesia and akinesia, limb rigidity, postural abnormalities, loss of vocalisation and blink reflex, and, on occasions, postural tremor. Measurement of caudate-putamen monoamine content at this time showed a profound loss in 3,4-dihydroxyphenylethylamine, homovanillic acid, and 3,4-dihydroxyphenylacetic acid concentrations. Measurement of neuropeptide concentrations in the caudate-putamen, internal and external segments of the globus pallidus, nucleus accumbens, substantia nigra, frontal cortex, and hippocampus showed met-enkephalin, leu-enkephalin, and cholecystokinin (CCK-8) concentrations to be unaffected by MPTP treatment. There was a small decrease in the substance P content of frontal cortex, but otherwise the content of this neuropeptide was unaltered. Parkinsonism in the marmoset, induced by MPTP treatment 10 days earlier, does not alter neuropeptide concentrations in the manner observed in Parkinson's disease.
 
Article
Cultured cerebellar astrocytes rapidly accumulate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) from the incubation medium, reaching a plateau within 10 min, whereas within that time negligible amounts of 1-methyl-4-phenylpyridinium (MPP+) have entered the astrocytes. MPTP accumulation is essentially independent of temperature and is proportional to extracellular concentration at steady state: The steady-state concentration achieved within these cells is about 50-fold higher at relatively low extracellular concentrations. MPTP appears to accumulate intracellularly within lysosomes, because lysosomotropic agents such as ammonium chloride and chloroquine markedly diminish the accumulation. Moreover, a proton gradient is required, because MPTP accumulation is abolished by the hydrogen ion antiporter monensin. Over an interval of several days, MPTP is converted to MPP+ intracellularly, with a concomitant decrease in medium MPTP and increase in medium MPP+. A constant, small but significant amount of MPP+ is retained intracellularly over a 72-h interval. Increasing the medium MPTP concentrations results in increased conversion of MPTP and enhanced intracellular retention of MPTP and MPP+. Neither MPTP nor MPP+ is neurotoxic to cultured cerebellar astrocytes as determined by cell counts and rate of conversion of MPTP to MPP+.
 
Article
The in vitro development of monoamine oxidase (MAO) activity and [3H]dopamine (DA) uptake capacity of dissociated cell cultures from rat embryo mesencephalon were correlated with the potency of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridine (MPP+) neurotoxicity. Specific activities of both MAO-A and MAO-B increased during in vitro development of the cultures, with MAO-B activity increasing 20-fold between the first and fourth week. Similarly, [3H]DA accumulation increased 2.6-fold between the first and third week in vitro, when it reached a plateau. Unexpectedly, the toxicities of MPTP and MPP+ were substantially decreased in the older cultures. Exposure to MPTP reduced [3H]DA accumulation per culture by 77% in 1-week-old cultures and by 36% in 4-week-old cultures. Similarly, damage caused by MPPT was reduced from 84% of control in the first week to 34% of control in the fourth week. The attenuation of neurotoxicity was not due to an increase in storage of MPP+ in the synaptic vesicles of DA neurons, nor to a change in the distribution of MPP+ between dopaminergic and other cellular components of the cultures. The damage to DA neurons caused by the mitochondrial toxin, rotenone, also showed a similar reduction in the older cultures. These observations coupled with an increase in lactate formation and glucose consumption during the in vitro development of the cultures suggest a shift toward increased glycolysis and decreased dependence on aerobic metabolism. This would render the cells more resistant to the inhibition of mitochondrial function by MPP+.
 
Top-cited authors
Mark P Mattson
  • Johns Hopkins University
George Perry
  • University of Texas at San Antonio
Barry Halliwell
  • National University of Singapore
D. Allan Butterfield
  • University of Kentucky
Xiongwei Zhu
  • Case Western Reserve University