Phosphatidylinositide 3-kinase and protein kinase C zeta mediate retinoic acid induction of DARPP-32 in medium size spiny neurons in vitro

Mature striatal medium size spiny neurons express the dopamine and cAMP-regulated phosphoprotein, 32 kDa (DARPP-32), but little is known about the mechanisms regulating its levels, or the specification of fully differentiated neuronal subtypes. Cell extrinsic molecules that increase DARPP-32 mRNA and/or protein levels include retinoic acid (RA), brain-derived neurotrophic factor, and estrogen (E₂). We now demonstrate that RA regulates DARPP-32 mRNA and protein in primary striatal neuronal cultures. Furthermore, DARPP-32 induction by RA in vitro requires phosphatidylinositide 3-kinase, but is independent of tropomyosin-related kinase B, cyclin-dependent kinase 5, and protein kinase B. Using pharmacologic inhibitors of various isoforms of protein kinase C (PKC), we also demonstrate that DARPP-32 induction by RA in vitro is dependent on PKC zeta (PKCζ). Thus, the signal transduction pathways mediated by RA are very different than those mediating DARPP-32 induction by brain-derived neurotrophic factor. These data support the presence of multiple signal transduction pathways mediating expression of DARPP-32 in vitro , including a novel, important pathway via which phosphatidylinositide 3-kinase regulates the contribution of PKCζ.     

Triazoloquinazolines as a novel class of phosphodiesterase 10A (PDE10A) inhibitors

Novel triazoloquinazolines have been found as phosphodiesterase 10A (PDE10A) inhibitors. Structure-activity studies improved the initial micromolar potency which was found in the lead compound by a 100-fold identifying 5-(1H-benzoimidazol-2-ylmethylsulfanyl)-2-methyl-[1,2,4]triazolo[1,5-c]quinazoline, 42 (PDE10A IC₅₀ = 12 nM) as the most potent compound from the series. Two X-ray structures revealed novel binding modes to the catalytic site of the PDE10A enzyme.     

Phosphodiesterase 10A inhibition modulates the sensitivity of the mesolimbic dopaminergic system to d-amphetamine: involvement of the D1-regulated feedback control of midbrain dopamine neurons

Phosphodiesterase (PDE) 10A is highly expressed in medium spiny neurons of the striatum, at the confluence of the corticostriatal glutamatergic and the midbrain dopaminergic pathways, both believed to be involved in the physiopathology of schizophrenia. There is a growing body of evidence suggesting that targeting PDE10A may be beneficial for the treatment of positive symptoms in schizophrenia. The aim of the present study was to investigate how PDE10A inhibition modulates mesolimbic dopaminergic neurotransmission. We found that the selective PDE10A inhibitor, MP-10, blocked d -amphetamine-induced hyperactivity as well as d -amphetamine-induced dopamine efflux in the nucleus accumbens in a dose-dependent manner. We further investigated the mechanism by which PDE10A inhibition affects dopaminergic neurotransmission. We report that MP-10 potentiated the effect of a high but not a low dose of d -amphetamine on the mean firing rate of dopaminergic neurons recorded from the ventral tegmental area. Similarly, the effect of a high, but not a low dose of d -amphetamine, was completely reversed by the selective D₁ antagonist, SCH23390. These data suggest that the D₁-regulated feedback control of midbrain dopamine neurons is a critical pathway involved in the modulation of the response of mesolimbic dopamine neurons to d -amphetamine by PDE10A inhibition.     

Novel 2-methoxyacylhydrazones as potent, selective PDE10A inhibitors with activity in animal models of schizophrenia

A series of 2-methoxyacylhydrazones were optimized to yield compounds with high affinity for PDE10A. Several compounds demonstrated efficacy in animal models of schizophrenia, including conditioned avoidance response and a pro-psychotic phencyclidine hyperactivity model.     

Adenosine A 2A receptor antagonists prevent the increase in striatal glutamate levels induced by glutamate uptake inhibitors

Active uptake by neurons and glial cells is the main mechanism for maintaining extracellular glutamate at low, non-toxic concentrations. Activation of adenosine A(2A) receptors increases extracellular glutamate levels, while A(2A) receptor antagonists reduce stimulated glutamate outflow. Whether a modulation of the glutamate uptake system is involved in the effects elicited by A(2A) receptor blockers has never been investigated. This study examined the ability of adenosine A(2A) receptor antagonists to prevent the increase in glutamate levels induced by blockade of the glutamate uptake. In rats implanted with a microdialysis probe in the dorsal striatum, perfusion with 4 mm l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC, a transportable competitive inhibitor of glutamate uptake), or 10 mm dihydrokainic acid (DHK, a non-transportable competitive inhibitor that mainly blocks the glial glutamate transporter GLT-1), significantly increased extracellular glutamate levels. The effects of PDC and DHK were completely prevented by the adenosine A(2A) receptor antagonists SCH 58261 (0.01 mg/kg i.p.) and/or ZM 241385 (5 nm via probe). Since an impairment in glutamate transporter function is thought to play a major role in neurodegenerative disorders, the regulation of glutamate uptake may be one of the mechanisms of the neuroprotective effects of A(2A) receptor antagonists.     

Synthesis and SAR study of new phenylimidazole-pyrazolo[1,5-c]quinazolines as potent phosphodiesterase 10A inhibitors

A series of phenylimidazole-pyrazolo[1,5-c]quinazolines 1a-q was designed, synthesized and characterised as a novel class of potent phophodiesterase 10A (PDE10A) inhibitors. In this series, 2,9-dimethyl-5-(2-(1-methyl-4-phenyl-1H-imidazol-2-yl)ethyl)pyrazolo[1,5-c]quinazoline (1q) showed the highest affinity for PDE10A enzyme (IC₅₀ = 16 nM).     

Differential phosphodiesterase expression and cytosolic Ca2+ in human CNS tumour cells and in non-malignant and malignant cells of rat origin

A promising attempt in the field of tumour therapy is the modulation of intracellular, proliferation-associated signalling pathways. The role of cyclic nucleotide phosphodiesterases (PDEs), key enzymes in cAMP/cGMP signal transduction, was investigated in two human CNS tumour cell lines as well as in the rat glioblastoma cell line C6 in comparison with rat cerebellar astrocytes with the emphasis on target evaluation. We found differential PDE expression patterns in human CNS tumour cell lines as well as in CNS cells of rat origin. In human glioblastoma cells, intracellular cAMP and Ca(2+) levels correlated well with the PDE expression pattern. There were, however, marked differences in PDE expression and Ca(2+) kinetics between the human glioblastoma cell lines. In contrast to human epithelial tumour cells, shown earlier by us to express significantly enhanced cAMP-specific PDE activity, this was not the case in rat glioblastoma cells compared with non-malignant rat astrocytes. Despite different levels of PDE1 and PDE4 expression and activity, cyclic nucleotide and Ca(2+) levels in non-malignant and malignant rat CNS cells were similar. These in vitro data do not support the concept of PDE1C representing a target exploitable for drug treatment of malignant CNS tumours.     

Pre-synaptic adenosine A2A receptors control cannabinoid CB1 receptor-mediated inhibition of striatal glutamatergic neurotransmission

An interaction between adenosine A(2A) receptors (A(2A) Rs) and cannabinoid CB(1) receptors (CB(1) Rs) has been consistently reported to occur in the striatum, although the precise mechanisms are not completely understood. As both receptors control striatal glutamatergic transmission, we now probed the putative interaction between pre-synaptic CB(1) R and A(2A) R in the striatum. In extracellular field potentials recordings in corticostriatal slices from Wistar rats, A(2A) R activation by CGS21680 inhibited CB(1) R-mediated effects (depression of synaptic response and increase in paired-pulse facilitation). Moreover, in superfused rat striatal nerve terminals, A(2A) R activation prevented, while A(2A) R inhibition facilitated, the CB(1) R-mediated inhibition of 4-aminopyridine-evoked glutamate release. In summary, the present study provides converging neurochemical and electrophysiological support for the occurrence of a tight control of CB(1) R function by A(2A) Rs in glutamatergic terminals of the striatum. In view of the key role of glutamate to trigger the recruitment of striatal circuits, this pre-synaptic interaction between CB(1) R and A(2A) R may be of relevance for the pathogenesis and the treatment of neuropsychiatric disorders affecting the basal ganglia.     

Co-localization and functional interaction between adenosine A(2A) and metabotropic group 5 receptors in glutamatergic nerve terminals of the rat striatum

The anti-Parkinsonian effect of glutamate metabotropic group 5 (mGluR5) and adenosine A(2A) receptor antagonists is believed to result from their ability to postsynaptically control the responsiveness of the indirect pathway that is hyperfunctioning in Parkinson's disease. mGluR5 and A(2A) antagonists are also neuroprotective in brain injury models involving glutamate excitotoxicity. Thus, we hypothesized that the anti-Parkinsonian and neuroprotective effects of A(2A) and mGluR5 receptors might be related to their control of striatal glutamate release that actually triggers the indirect pathway. The A(2A) agonist, CGS21680 (1-30 nM) facilitated glutamate release from striatal nerve terminals up to 57%, an effect prevented by the A(2A) antagonist, SCH58261 (50 nM). The mGluR5 agonist, CHPG (300-600 mum) also facilitated glutamate release up to 29%, an effect prevented by the mGluR5 antagonist, MPEP (10 microm). Both mGluR5 and A(2A) receptors were located in the active zone and 57 +/- 6% of striatal glutamatergic nerve terminals possessed both A(2A) and mGluR5 receptors, suggesting a presynaptic functional interaction. Indeed, submaximal concentrations of CGS21680 (1 nM) and CHPG (100 microm) synergistically facilitated glutamate release and the facilitation of glutamate release by 10 nM CGS21680 was prevented by 10 microm MPEP, whereas facilitation by 300 microm CHPG was prevented by 10 nM SCH58261. These results provide the first direct evidence that A(2A) and mGluR5 receptors are co-located in more than half of the striatal glutamatergic terminals where they facilitate glutamate release in a synergistic manner. This emphasizes the role of the modulation of glutamate release as a likely mechanism of action of these receptors both in striatal neuroprotection and in Parkinson's disease.     

A further study on the regulation of cyclic nucleotide phosphodiesterase activity in neuroblastoma cells: Effect of growth

Summary Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) phosphodiesterase activity in mouse neuroblastoma cells in culture markedly increased during exponential growth and reached a maximal level at confluency; whereas guanosine 3′, 5′-cyclic monophosphate (cyclic GMP) phosphodiesterase activity only slightly but significantly increased under a similar experimental condition. The increase in cyclic AMP phosphodiesterase activity was blocked by both cycloheximide and dactinomycin, whereas the increase in cyclic GMP phosphodiesterase was blocked by only cycloheximide. When the confluent cells were replated at low density, the cyclic nucleotide phosphodiesterase activity decreased; however, when they were plated at high cell density which equaled confluency, the enzyme activity did not decrease. Unlike cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity did not change significantly in prostaglandin E₁-treated cells, but decreased in cells treated with the inhibitor of phosphodiesterase. Like cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity also did not change in cells treated with serum-free medium, X-irradiation, sodium butyrate and 6-thioguanine. This work was supported by USPHS NS-09230, and DRG-1273 from Damon Runyon-Walter Winchell Cancer Fund.     

l-DOPA activates ERK signaling and phosphorylates histone H3 in the striatonigral medium spiny neurons of hemiparkinsonian mice

In the dopamine-depleted striatum, extracellular signal-regulated kinase (ERK) signaling is implicated in the development of l -DOPA-induced dyskinesia. To gain insights on its role in this disorder, we examined the effects of l -DOPA on the state of phosphorylation of ERK and downstream target proteins in striatopallidal and striatonigral medium spiny neurons (MSNs). For this purpose, we employed mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoters for the dopamine D₂ receptor ( Drd2 -EGFP mice) or the dopamine D₁ receptor ( Drd1a -EGFP mice), which are expressed in striatopallidal and striatonigral MSNs, respectively. In 6-hydroxydopamine-lesioned Drd2 -EGFP mice, l -DOPA increased the phosphorylation of ERK, mitogen- and stress-activated kinase 1 and histone H3, selectively in EGFP-negative MSNs. Conversely, a complete co-localization between EGFP and these phosphoproteins was observed in Drd1a -EGFP mice. The effect of l -DOPA was prevented by blockade of dopamine D₁ receptors. The same pattern of activation of ERK signaling was observed in dyskinetic mice, after repeated administration of l -DOPA. Our results demonstrate that in the dopamine-depleted striatum, l -DOPA activates ERK signaling specifically in striatonigral MSNs. This regulation may result in ERK-dependent changes in striatal plasticity leading to dyskinesia.     

Comparison of plate count agar and R2A medium for enumeration of heterotrophic bacteria in natural mineral water

In order to recover as many viable bacteria as possible from natural mineral water, in this study we have compared the counts obtained with the standard method (pour plate procedure with Plate Count Agar (PCA)) and counts with alternative test methods (PCA/spread plates, R2A medium/pour plates and R2A medium/spread plates). The results showed that counts with R2A medium/spread plates at 22°C and after a 7-day incubation period were more than 343% higher than those obtained with PCA/pour plate method. At 37°C and after a 3-day incubation period, the R2A pour plate technique gave counts about 368% greater than for the standard method. Moreover, while Pseudomonas, Comamonas and Acinetobacter species were isolated both from PCA and R2A medium, Flavobacterium spp. and Arthrobacter spp. were isolated only from R2A medium. For its higher productivity, R2A medium should be recommended for heterotrophic plate counts in natural mineral water.     

Polyamine-phospholipid interaction probed by the accessibility of the phospholipidsn-2 ester bond to the action of phospholipase A2

Summary Conditions were used where the action of porcine pancreatic phospholipase A2 on phospholipids can be followed in the absence of added calcium and the catalytic activity is supported by the calcium brought with the nanomolar enzyme. Therefore, alterations in the enzyme velocity resulting from the presence of spermine or spermidine could be specifically studied using 1-palmitoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine (PPHPC) and 1-palmitoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphoglycerol (PPHPG) as substrates. Both spermine and spermidine activated the hydrolysis of PPHPG fourfold at polyamine/phospholipid molar ratios of approximately 11 and 121, respectively. Double-reciprocal plots of enzyme activityvs. PPHPG concentration revealed the enhancement to be due to increased apparentV _max while the apparentK _m was slightly increased. In the presence of 4mm CaCl₂ inhibition by polyamines of PPHPG hydrolysis by phospholipase A2 was observed. Using synthetic diamines we could further demonstrate that two primary amino groups are required for the activation. In the absence of exogenous CaCl₂ polyamines inhibited the hydrolysis of PPHPC by phospholipase A2. The presence of 4mm CaCl₂ reversed this inhibition and a twofold activation was observed at 10 m spermine. The results obtained indicate that the activation of PLA2 by spermine and spermidine is produced at the level of the substrate, PPHPG. This implies the formation of complexes of phosphatidylglycerol and polyamines with defined stoichiometries.     

Adenosine A2A receptors inhibit the conductance of NMDA receptor channels in rat neostriatal neurons

Summary Whole-cell patch clamp experiments were carried out in rat striatal brain slices. In a subset of striatal neurons (70–80%), NMDA-induced inward currents were inhibited by the adenosine AZA receptor selective agonist CGS 21680. The non-selective adenosine receptor antagonist 8-(p-sulphophenyl)-theophylline and the AZA receptor selective antagonist 8-(3chlorostyryl) caffeine abolished the inhibitory action of CGS 21680. Intracellular GDP--S, which is known to prevent G protein-mediated reactions, also eliminated the effect of CGS 21680. Extracellular dibutyryl cAMP, a membrane permeable analogue of cAMP, and intracellular Sp-cAMPS, an activator of cAMP-dependent protein kinases (PKA), both abolished the CGS 21680-induced inhibition. By contrast, Rp-cAMPS and PKI 14–24 amide, two inhibitors of PKA had no effect. Intracellular U-73122 (a phospholipase C inhibitor) and heparin (an inositoltriphosphate antagonist) prevented the effect of CGS 21680. Finally, a more efficient buffering of intracellular Ca²⁺ by a substitution of EGTA (11 mM) by BAPTA (5.5 mM) acted like U-73122 or heparin. Hence, AZA receptors appear to negatively modulate NMDA receptor channel conductance via the phospholipase C/inositoltriphosphate/Ca²⁺ pathway rather than the adenylate cyclase/PKA pathway.     

Expression of the 2',3'-cAMP-adenosine pathway in astrocytes and microglia

Many organs express the extracellular 3',5'-cAMP-adenosine pathway (conversion of extracellular 3',5'-cAMP to 5'-AMP and 5'-AMP to adenosine). Some organs release 2',3'-cAMP (isomer of 3',5'-cAMP) and convert extracellular 2',3'-cAMP to 2'- and 3'-AMP and convert these AMPs to adenosine (extracellular 2',3'-cAMP-adenosine pathway). As astrocytes and microglia are important participants in the response to brain injury and adenosine is an endogenous neuroprotectant, we investigated whether these extracellular cAMP-adenosine pathways exist in these cell types. 2',3'-, 3',5'-cAMP, 5'-, 3'-, and 2'-AMP were incubated with mouse primary astrocytes or primary microglia for 1 h and purine metabolites were measured in the medium by mass spectrometry. There was little evidence of a 3',5'-cAMP-adenosine pathway in either astrocytes or microglia. In contrast, both cell types converted 2',3'-cAMP to 2'- and 3'-AMP (with 2'-AMP being the predominant product). Although both cell types converted 2'- and 3'-AMP to adenosine, microglia were five- and sevenfold, respectively, more efficient than astrocytes in this regard. Inhibitor studies indicated that the conversion of 2',3'-cAMP to 2'-AMP was mediated by a different ecto-enzyme than that involved in the metabolism of 2',3'-cAMP to 3'-AMP and that although CD73 mediates the conversion of 5'-AMP to adenosine, an alternative ecto-enzyme metabolizes 2'- or 3'-AMP to adenosine.     

Regulation of Sarcoplasmic Reticulum Ca2+ ATPase 2 (SERCA2) Activity by Phosphodiesterase 3A (PDE3A) in Human Myocardium: PHOSPHORYLATION-DEPENDENT INTERACTION OF PDE3A1 WITH SERCA2*

Cyclic nucleotide phosphodiesterase 3A (PDE3) regulates cAMP-mediated signaling in the heart, and PDE3 inhibitors augment contractility in patients with heart failure. Studies in mice showed that PDE3A, not PDE3B, is the subfamily responsible for these inotropic effects and that murine PDE3A1 associates with sarcoplasmic reticulum Ca²⁺ ATPase 2 (SERCA2), phospholamban (PLB), and AKAP18 in a multiprotein signalosome in human sarcoplasmic reticulum (SR). Immunohistochemical staining demonstrated that PDE3A co-localizes in Z-bands of human cardiac myocytes with desmin, SERCA2, PLB, and AKAP18. In human SR fractions, cAMP increased PLB phosphorylation and SERCA2 activity; this was potentiated by PDE3 inhibition but not by PDE4 inhibition. During gel filtration chromatography of solubilized SR membranes, PDE3 activity was recovered in distinct high molecular weight (HMW) and low molecular weight (LMW) peaks. HMW peaks contained PDE3A1 and PDE3A2, whereas LMW peaks contained PDE3A1, PDE3A2, and PDE3A3. Western blotting showed that endogenous HMW PDE3A1 was the principal PKA-phosphorylated isoform. Phosphorylation of endogenous PDE3A by rPKAc increased cAMP-hydrolytic activity, correlated with shift of PDE3A from LMW to HMW peaks, and increased co-immunoprecipitation of SERCA2, cav3, PKA regulatory subunit (PKARII), PP2A, and AKAP18 with PDE3A. In experiments with recombinant proteins, phosphorylation of recombinant human PDE3A isoforms by recombinant PKA catalytic subunit increased co-immunoprecipitation with rSERCA2 and rat rAKAP18 (recombinant AKAP18). Deletion of the recombinant human PDE3A1/PDE3A2 N terminus blocked interactions with recombinant SERCA2. Serine-to-alanine substitutions identified Ser-292/Ser-293, a site unique to human PDE3A1, as the principal site regulating its interaction with SERCA2. These results indicate that phosphorylation of human PDE3A1 at a PKA site in its unique N-terminal extension promotes its incorporation into SERCA2/AKAP18 signalosomes, where it regulates a discrete cAMP pool that controls contractility by modulating phosphorylation-dependent protein-protein interactions, PLB phosphorylation, and SERCA2 activity.     

Genetic and pharmacological inactivation of the adenosine A2A receptor attenuates 3-nitropropionic acid-induced striatal damage

Adenosine A2A receptor (A2AR) antagonism attenuates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration and quinolinic acid-induced excitotoxicity in the neostriatum. As A2ARs are enriched in striatum, we investigated the effect of genetic and pharmacological A2A inactivation on striatal damage produced by the mitochondrial complex II inhibitor 3-nitropriopionic acid (3-NP). 3-NP was administered to A2AR knockout (KO) and wild-type (WT) littermate mice over 5 days. Bilateral striatal lesions were analyzed from serial brain tissue sections. Whereas all of the 3-NP-treated WT mice (C57BL/6 genetic background) had bilateral striatal lesions, only one of eight of the 3-NP-treated A2AR KO mice had detectable striatal lesions. Similar attenuation of 3-NP-induced striatal damage was observed in A2AR KO mice in a 129-Steel background. In addition, the effect of pharmacological antagonism on 3-NP-induced striatal neurotoxicity was tested by pre-treatment of C57Bl/6 mice with the A2AR antagonist 8-(3-chlorostyryl) caffeine (CSC). Although bilateral striatal lesions were observed in all mice treated either with 3-NP alone or 3-NP plus vehicle, there were no demonstrable striatal lesions in mice treated with CSC (5 mg/kg) plus 3-NP and in five of six mice treated with CSC (20 mg/kg) plus 3-NP. We conclude that both genetic and pharmacological inactivation of the A2AR attenuates striatal neurotoxicity produced by 3-NP. Since the clinical and neuropathological features of 3-NP-induced striatal damage resemble those observed in Huntington's disease, the results suggest that A2AR antagonism may be a potential therapeutic strategy in Huntington's disease patients.     

S100A10‐Mediated Translocation of Annexin‐A2 to SNARE Proteins in Adrenergic Chromaffin Cells Undergoing Exocytosis

In neuroendocrine cells, annexin‐A2 is implicated as a promoter of monosialotetrahexosylganglioside (GM1)‐containing lipid microdomains that are required for calcium‐regulated exocytosis. As soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) require a specific lipid environment to mediate granule docking and fusion, we investigated whether annexin‐A2‐induced lipid microdomains might be linked to the SNAREs present at the plasma membrane. Stimulation of adrenergic chromaffin cells induces the translocation of cytosolic annexin‐A2 to the plasma membrane, where it colocalizes with SNAP‐25 and S100A10. Cross‐linking experiments performed in stimulated chromaffin cells indicate that annexin‐A2 directly interacts with S100A10 to form a tetramer at the plasma membrane. Here, we demonstrate that S100A10 can interact with vesicle‐associated membrane protein 2 (VAMP2) and show that VAMP2 is present at the plasma membrane in resting adrenergic chromaffin cells. Tetanus toxin that cleaves VAMP2 solubilizes S100A10 from the plasma membrane and inhibits the translocation of annexin‐A2 to the plasma membrane. Immunogold labelling of plasma membrane sheets combined with spatial point pattern analysis confirmed that S100A10 is present in VAMP2 microdomains at the plasma membrane and that annexin‐A2 is observed close to S100A10 and to syntaxin in stimulated chromaffin cells. In addition, these results showed that the formation of phosphatidylinositol (4,5)‐bisphosphate (PIP₂) microdomains colocalized with S100A10 in the vicinity of docked granules, suggesting a functional interplay between annexin‐A2‐mediated lipid microdomains and SNAREs during exocytosis.