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• W2012249786 abstract "Phosphodiesterase 10A (PDE10A) is a dual substrate PDE that can hydrolyze both cGMP and cAMP. In brain, PDE10A is almost exclusively expressed in the striatum. In several studies, PDE10A has been implicated in regulation of striatal output using either specific inhibitors or PDE10A knock-out mice and has been suggested as a promising target for novel antipsychotic drugs. In striatal medium spiny neurons, PDE10A is localized at the plasma membrane and in dendritic spines close to postsynaptic densities. In the present study, we identify PDE10A as the major cAMP PDE in mouse striatum and monitor PKA-dependent PDE10A phosphorylation. With recombinantly expressed PDE10A we demonstrate that phosphorylation does not alter PDE10A activity. In striatum, PDE10A was found to be associated with the A kinase anchoring protein AKAP150 suggesting the existence of a multiprotein signaling complex localizing PDE10A to a specific functional context at synaptic membranes. Furthermore, the cAMP effector PKA, the NMDA receptor subunits NR2A and -B, as well as PSD95, were tethered to the complex. In agreement, PDE10A was almost exclusively found in multiprotein complexes as indicated by migration in high molecular weight fractions in size exclusion chromatography. Finally, affinity of PDE10A to the signaling complexes formed around AKAP150 was reduced by PDE10A phosphorylation. The data indicate that phosphorylation of PDE10 has an impact on the interaction with other signaling proteins and adds an additional line of complexity to the role of PDE10 in regulation of synaptic transmission.Background: In striatum, cortical glutamatergic and midbrain dopaminergic inputs are integrated via cAMP.Results: PDE10A, the major cAMP-hydrolyzing enzyme in striatum, is targeted into a signaling complex containing the scaffolding proteins AKAP150, PSD95, and the NMDA receptor and released upon phosphorylation.Conclusion: Targeting of PDE10 is under control of cAMP/PKA activity.Significance: Phosphorylation-dependent release of PDE10 gives rise to a feed forward mechanism. Phosphodiesterase 10A (PDE10A) is a dual substrate PDE that can hydrolyze both cGMP and cAMP. In brain, PDE10A is almost exclusively expressed in the striatum. In several studies, PDE10A has been implicated in regulation of striatal output using either specific inhibitors or PDE10A knock-out mice and has been suggested as a promising target for novel antipsychotic drugs. In striatal medium spiny neurons, PDE10A is localized at the plasma membrane and in dendritic spines close to postsynaptic densities. In the present study, we identify PDE10A as the major cAMP PDE in mouse striatum and monitor PKA-dependent PDE10A phosphorylation. With recombinantly expressed PDE10A we demonstrate that phosphorylation does not alter PDE10A activity. In striatum, PDE10A was found to be associated with the A kinase anchoring protein AKAP150 suggesting the existence of a multiprotein signaling complex localizing PDE10A to a specific functional context at synaptic membranes. Furthermore, the cAMP effector PKA, the NMDA receptor subunits NR2A and -B, as well as PSD95, were tethered to the complex. In agreement, PDE10A was almost exclusively found in multiprotein complexes as indicated by migration in high molecular weight fractions in size exclusion chromatography. Finally, affinity of PDE10A to the signaling complexes formed around AKAP150 was reduced by PDE10A phosphorylation. The data indicate that phosphorylation of PDE10 has an impact on the interaction with other signaling proteins and adds an additional line of complexity to the role of PDE10 in regulation of synaptic transmission. Background: In striatum, cortical glutamatergic and midbrain dopaminergic inputs are integrated via cAMP. Results: PDE10A, the major cAMP-hydrolyzing enzyme in striatum, is targeted into a signaling complex containing the scaffolding proteins AKAP150, PSD95, and the NMDA receptor and released upon phosphorylation. Conclusion: Targeting of PDE10 is under control of cAMP/PKA activity. Significance: Phosphorylation-dependent release of PDE10 gives rise to a feed forward mechanism. To date, 11 families of phosphodiesterases (PDE1 to PDE11) 2The abbreviations used are: PDEphosphodiesteraseAKAPA-kinase anchor proteinMP-102-[4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)-phenoxymethyl]-quinolinePP2Aprotein phosphatase 2A. have been identified that are differentially expressed in mammalian tissues and cells (1.Bender A.T. Beavo J.A. Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use.Pharmacol. Rev. 2006; 58: 488-520Crossref PubMed Scopus (1430) Google Scholar). Phosphodiesterases tightly regulate cyclic nucleotide signaling by hydrolysis of the second messengers cGMP and cAMP. In the central nervous system, comparatively high cyclic nucleotide-degrading activity is found. Here, many neurons express multiple PDEs with different subcellular localization and/or substrate specificities. phosphodiesterase A-kinase anchor protein 2-[4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)-phenoxymethyl]-quinoline protein phosphatase 2A. PDE10 was initially discovered in brain and is capable of hydrolyzing both second messengers, cAMP and cGMP, but has higher affinity for cAMP (2.Fujishige K. Kotera J. Michibata H. Yuasa K. Takebayashi S. Okumura K. Omori K. Cloning and characterization of a novel human phosphodiesterase that hydrolyzes both cAMP and cGMP (PDE10A).J. Biol. Chem. 1999; 274: 18438-18445Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 3.Loughney K. Snyder P.B. Uher L. Rosman G.J. Ferguson K. Florio V.A. Isolation and characterization of PDE10A, a novel human 3′,5′-cyclic nucleotide phosphodiesterase.Gene. 1999; 234: 109-117Crossref PubMed Scopus (199) Google Scholar, 4.Soderling S.H. Bayuga S.J. Beavo J.A. Isolation and characterization of a dual-substrate phosphodiesterase gene family: PDE10A.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 7071-7076Crossref PubMed Scopus (357) Google Scholar). PDE10 contains a tandem of regulatory GAF domains. cAMP binds to the second of these GAF domains, GAF-B (5.Gross-Langenhoff M. Hofbauer K. Weber J. Schultz A. Schultz J.E. cAMP is a ligand for the tandem GAF domain of human phosphodiesterase 10 and cGMP for the tandem GAF domain of phosphodiesterase 11.J. Biol. Chem. 2006; 281: 2841-2846Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), and activates the enzyme at least 3-fold (6.Jäger R. Russwurm C. Schwede F. Genieser H.-G. Koesling D. Russwurm M. Activation of PDE10 and PDE11 phosphodiesterases.J. Biol. Chem. 2012; 287: 1210-1219Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). PDE10 is encoded by one gene (PDE10A) that gives rise to several splice variants (7.Fujishige K. Kotera J. Yuasa K. Omori K. The human phosphodiesterase PDE10A gene genomic organization and evolutionary relatedness with other PDEs containing GAF domains.Eur. J. Biochem. 2000; 267: 5943-5951Crossref PubMed Scopus (47) Google Scholar, 8.O'Connor V. Genin A. Davis S. Karishma K.K. Doyère V. De Zeeuw C.I. Sanger G. Hunt S.P. Richter-Levin G. Mallet J. Laroche S. Bliss T.V. French P.J. Differential amplification of intron-containing transcripts reveals long term potentiation-associated up-regulation of specific Pde10A phosphodiesterase splice variants.J. Biol. Chem. 2004; 279: 15841-15849Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) of which PDE10A2 appears to be the major neuronal form in various species. The highest expression of PDE10A has been detected in the striatum (6.Jäger R. Russwurm C. Schwede F. Genieser H.-G. Koesling D. Russwurm M. Activation of PDE10 and PDE11 phosphodiesterases.J. Biol. Chem. 2012; 287: 1210-1219Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 9.Fujishige K. Kotera J. Omori K. Striatum- and testis-specific phosphodiesterase PDE10A isolation and characterization of a rat PDE10A.Eur. J. Biochem. 1999; 266: 1118-1127Crossref PubMed Scopus (173) Google Scholar) where it is restricted to the GABAergic medium spiny neurons (10.Xie Z. Adamowicz W.O. Eldred W.D. Jakowski A.B. Kleiman R.J. Morton D.G. Stephenson D.T. Strick C.A. Williams R.D. Menniti F.S. Cellular and subcellular localization of PDE10A, a striatum-enriched phosphodiesterase.Neuroscience. 2006; 139: 597-607Crossref PubMed Scopus (179) Google Scholar, 11.Nishi A. Kuroiwa M. Miller D.B. O'Callaghan J.P. Bateup H.S. Shuto T. Sotogaku N. Fukuda T. Heintz N. Greengard P. Snyder G.L. Distinct roles of PDE4 and PDE10A in the regulation of cAMP/PKA signaling in the striatum.J. Neurosci. 2008; 28: 10460-10471Crossref PubMed Scopus (227) Google Scholar, 12.Sano H. Nagai Y. Miyakawa T. Shigemoto R. Yokoi M. Increased social interaction in mice deficient of the striatal medium spiny neuron-specific phosphodiesterase 10A2.J. Neurochem. 2008; 105: 546-556Crossref PubMed Scopus (84) Google Scholar). These neurons represent ∼95% of striatal neurons and integrate cortical glutamatergic input and midbrain dopaminergic signaling. They form the major input station for the basal ganglia that are involved in planning and modulation of movement pathways and a variety of other cognitive processes. Dysfunction of striatal circuitry is implicated in the pathophysiology of many brain disorders such as Parkinson disease, Huntington disease, schizophrenia, and substance abuse (13.Kapur S. How antipsychotics become anti-“psychotic”: from dopamine to salience to psychosis.Trends Pharmacol. Sci. 2004; 25: 402-406Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 14.Everitt B.J. Robbins T.W. Neural systems of reinforcement for drug addiction: from actions to habits to compulsion.Nat. Neurosci. 2005; 8: 1481-1489Crossref PubMed Scopus (2719) Google Scholar, 15.DeLong M.R. Wichmann T. Circuits and circuit disorders of the basal ganglia.Arch. Neurol. 2007; 64: 20-24Crossref PubMed Scopus (777) Google Scholar). The high expression of PDE10A in medium spiny neurons suggests an important role in modulation of striatal function through cAMP degradation and termination of cAMP/PKA signaling. Consequently, enhancement of cAMP signaling through PDE10A knock-out or pharmacological inhibition of PDE10A has been shown to affect locomotor activity and acquisition of conditioned avoidance (16.Siuciak J.A. McCarthy S.A. Chapin D.S. Fujiwara R.A. James L.C. Williams R.D. Stock J.L. McNeish J.D. Strick C.A. Menniti F.S. Schmidt C.J. Genetic deletion of the striatum-enriched phosphodiesterase PDE10A: evidence for altered striatal function.Neuropharmacology. 2006; 51: 374-385Crossref PubMed Scopus (215) Google Scholar17.Siuciak J.A. Chapin D.S. Harms J.F. Lebel L.A. McCarthy S.A. Chambers L. Shrikhande A. Wong S. Menniti F.S. Schmidt C.J. Inhibition of the striatum-enriched phosphodiesterase PDE10A: a novel approach to the treatment of psychosis.Neuropharmacology. 2006; 51: 386-396Crossref PubMed Scopus (249) Google Scholar, 18.Hebb A.L. Robertson H.A. Denovan-Wright E.M. Phosphodiesterase 10A inhibition is associated with locomotor and cognitive deficits and increased anxiety in mice.Eur. Neuropsychopharmacol. 2008; 18: 339-363Crossref PubMed Scopus (51) Google Scholar19.Schmidt C.J. Chapin D.S. Cianfrogna J. Corman M.L. Hajos M. Harms J.F. Hoffman W.E. Lebel L.A. McCarthy S.A. Nelson F.R. Proulx-LaFrance C. Majchrzak M.J. Ramirez A.D. Schmidt K. Seymour P.A. Siuciak J.A. Tingley 3rd, F.D. Williams R.D. Verhoest P.R. Menniti F.S. Preclinical characterization of selective phosphodiesterase 10A inhibitors: a new therapeutic approach to the treatment of schizophrenia.J. Pharmacol. Exp. Ther. 2008; 325: 681-690Crossref PubMed Scopus (252) Google Scholar). These results led to the proposal of PDE10A as a promising target for novel antipsychotic therapies. Here, we describe PDE10A as the major cAMP PDE in striatum. Interaction of PDE10A with a postsynaptic signaling complex is demonstrated by coprecipitation of PDE10 with the scaffold protein AKAP150, PKA as well as PSD95 and NMDA receptor subunits. Such a complex might put PDE10A into the position of a “gate keeper” that limits cAMP accumulation at postsynaptic sites, prevents spreading of synaptic signals into the cell body, and ensures precisely timed phosphorylation and thereby regulation of NMDA receptors. We demonstrate PKA-dependent phosphorylation of PDE10A in striatal slices and identify protein phosphatase 2A (PP2A) as the responsible phosphatase. Moreover, phosphorylation of PDE10A2 does not alter enzymatic properties but releases PDE10A from the identified signaling complex. All experiments were performed using 3-month-old male mice of the C57BL/6 strain. After sacrificing the animals, the brain was quickly removed and the striata from the left and right hemisphere were isolated in less than 2 min. The striata of two mice were immediately homogenized in 10 volumes (w/v) of ice-cold lysis buffer (50 mm NaCl, 1 mm EDTA, 2 mm dl-dithiothreitol, 50 mm triethanolamine/hydrochloride, pH 7.4, containing the protease inhibitors phenylmethylsulfonyl fluoride (0.4 mm), benzamidine (0.2 mm), and pepstatin A (1 μm)) using a glass-glass Potter-Elvehjem homogenizer. The lysate was cleared from nuclei and cellular debris by centrifugation at 800 × g (10 min, 4 °C) and then recentrifuged at 125,000 × g (40 min, 4 °C) to obtain the cytosolic fraction. Membranes were resuspended in the original volume of lysis buffer. Equal volumes of all fractions (corresponding to 20 μg of protein in the homogenate) were applied to SDS-PAGE. Synaptosomal cytosol and membranes were prepared as described (20.Russwurm M. Wittau N. Koesling D. Guanylyl cyclase/PSD-95 interaction: targeting of the nitric oxide-sensitive α2β1 guanylyl cyclase to synaptic membranes.J. Biol. Chem. 2001; 276: 44647-44652Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). HEK293 cells were grown in DMEM with 5% heat-inactivated fetal calf serum and 1% penicillin/streptomycin at 37 °C in a humidified 5% CO2 atmosphere. Mouse PDE10A2 was cloned into the pcDNA3.1 zeo+ vector via NheI/NotI sites and 8 μg of vector/75 cm2 bottle were transfected into HEK293 cells using the FuGENE6 protocol (Promega). Transfected cells were harvested 48–72 h post-transfection, washed twice with phosphate-buffered saline, resuspended in 0.5 ml of lysis buffer, and lysed by sonication (two 5-s pulses). Cellular debris was eliminated by centrifugation (800 × g, 10 min, 4 °C). The lysate was split for comparative analysis of phosphorylated versus non-phosphorylated PDE10A2 and preincubated for 10 min with 5 mm MgCl2, 0.5 mm ATP, 1 μm okadaic acid and PhosSTOP (Roche Applied Science) in lysis buffer. In vitro phosphorylation was performed by addition of the catalytic subunit of PKA (0.5 μg, Jena Bioscience) for 30 min at 37 °C. For non-phospho control, ATP and PKA were omitted. PDE10A2-expressing HEK293 cells were homogenized in cell extraction buffer (50 mm NaCl, 50 mm triethanolamine/hydrochloride, pH 7.4, containing 0.4 mm phenylmethylsulfonyl fluoride, 0.2 mm benzamidine, 1 μm pepstatin A, and 1 μm okadaic acid) by 15 strokes with a glass-glass Potter-Elvehjem homogenizer. The cell extract was diluted with PKA buffer (0.5 mm ATP, 3 mm MgCl2, 0.5 mg/ml of BSA, 2 mm dl-dithiothreitol, 50 mm triethanolamine/hydrochloride, pH 7.4, final concentrations) and incubated with or without 0.5 μg (800 units) of PKA (Jena Biosciences, PR-318) for 30 min at 37 °C. An aliquot of the phosphorylated sample was saved for Western blot before purification of pPDE10 using the Pierce phosphoprotein enrichment kit (Thermo Scientific) following the suppliers instructions as follows. The sample was diluted 10-fold with the supplied lysis/binding/wash buffer containing CHAPS (0.25%) and then incubated with the column for 1 h at 4 °C with gentle agitation. Flow-through was discarded and the phosphorylated protein was eluted with 1 ml of supplied elution buffer without CHAPS. All samples were applied to a 9% SDS gel. For detection, PDE10 antibody (6.Jäger R. Russwurm C. Schwede F. Genieser H.-G. Koesling D. Russwurm M. Activation of PDE10 and PDE11 phosphodiesterases.J. Biol. Chem. 2012; 287: 1210-1219Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) and pPDE10 antibody were used. The pPDE10 antibody was generated against phosphorylated amino acids 11–20 of PDE10A2 in rabbits and purified according to the manufacturer's protocol (PSL, Heidelberg, Germany). PDE activity was measured by the conversion of [32P]cyclic nucleotide monophosphate into [32P]nucleotide monophosphate as described previously (21.Jäger R. Schwede F. Genieser H.-G. Koesling D. Russwurm M. Activation of PDE2 and PDE5 by specific GAF ligands: delayed activation of PDE5.Br. J. Pharmacol. 2010; 161: 1645-1660Crossref PubMed Scopus (26) Google Scholar). Reaction mixtures contained 0.1 μl of the striatal or HEK cell homogenates. Substrate concentrations were 0.03 to 1 μm cAMP or 1 μm cGMP, as indicated. Data are mean ± S.E. of at least three independent experiments performed in duplicates. PDE10A2 activation by the GAF domain ligand 7-CH-cAMP (Biolog) was determined at a substrate concentration of 0.03 μm cAMP. To determine the dissociation rate of cAMP from the PDE10A2 GAF domain, PDE10A2 was preincubated with 3 mm MgCl2 and 10 μm cAMP for 2 min at 37 °C. PDE10A2 was then diluted 100-fold and PDE activity was determined at various time points between 0 and 32 min. Three-month-old male mice were killed, and brains were removed and placed in ice-cold Krebs-Henseleit (KH) buffer containing the following: 118 mm NaCl, 4.7 mm KCl, 2.55 mm CaCl2, 1.2 mm KH2PO4, 1.2 mm MgSO4, 25 mm NaHCO3, and 27.8 mm d-glucose equilibrated to pH 7.4 by aeration with 95% O2, 5% CO2. Brain slices (coronal, 300 μm thick) were cut using a Vibroslice (NVSLM1, World Precision Instruments). The striatum was isolated and immediately transferred to a nylon net submerged in KH buffer, kept at pH 7.4 by aeration as described above (room temperature). After 1 h of recovery, slices were transferred to incubation chambers containing 10 ml of KH, kept at 37 °C, and equilibrated for a further 30 min. Slices were preincubated with PDE and phosphatase inhibitors for 10 min as indicated, transferred to new incubation chambers containing NMDA with or without phosphatase inhibitors as indicated for an additional 5 min. Slices were instantly frozen in liquid nitrogen. For immunodetection, single slices were homogenized in 240 μl of lysis buffer as described above. SDS-PAGE sample buffer (60 μl, ×4) was immediately added and 20 μl of each sample was applied to electrophoresis. The striata of two mice were homogenized in 1 ml of ice-cold Nonidet P-40 buffer (150 mm NaCl, 1.5 mm EDTA, 1% Nonidet P-40, 50 mm Tris, pH 7.4, containing protease inhibitors as above) using a glass-glass Potter-Elvehjem homogenizer. The lysate was cleared by centrifugation at 800 × g (10 min, 4 °C) and added to Dynabeads Protein A (Invitrogen) that were loaded with ∼50 μg of anti-PDE10A antibody (6.Jäger R. Russwurm C. Schwede F. Genieser H.-G. Koesling D. Russwurm M. Activation of PDE10 and PDE11 phosphodiesterases.J. Biol. Chem. 2012; 287: 1210-1219Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) per 10 mg of beads. Incubation was for 1 h at 4 °C in the absence or presence of the purified antigen (500 μg). Unspecifically bound proteins were removed by three washes with PBS and precipitated proteins were eluted by boiling in SDS sample buffer (30 μl/mg of Dynabeads) and loaded to SDS gels (15–20 μl per lane). One-tenth of the striatal lysate was applied for input loading. For immunoprecipitation of phosphorylated PDE10, striatal slices from 2 mice were prepared. Phosphorylation was induced by incubation of the slices with papaverine (100 μm) and forskolin (10 μm) for 10 min in the presence of phosphatase inhibitors (0.5 μm okadaic acid, 5 μm cyclosporin A, 0.2 μm calyculin A). Slices were pooled and instantly homogenized in 1 ml of Nonidet P-40 buffer (as above, but additionally containing phosphatase inhibitors) before precipitation by PDE10 antibody coupled to Dynabeads (see above). AKAP150 and the NMDA receptor subunit NR2A were precipitated from striata of 2 mice as described above using 10 μg of antibodies (AKAP150 sc-6445; NMDAϵ1 sc-1468, Santa Cruz) per 10 mg of beads. After washing, 55 μg of Dynabeads with bound proteins/sample were used in PDE assays. Measurements were performed in duplicates. Data represent mean ± S.E. of at least three independent experiments. Protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes (Protran BA-85, Schleicher & Schuell) using standard procedures. After blocking (Roti-Block; Carl Roth), proteins were detected using the following antibodies: PDE10A, AKAP150, and NMDAϵ1 (see above), NMDAϵ2 (sc-1469, Santa Cruz), PP2B (sc-6123, Santa Cruz), and PSD95 (MA1–046, Thermo Scientific). Anti-pPDE10A was generated in rabbits against the phosphorylated N-terminal peptide (amino acids 11–20). Purification of the antiserum was performed according to the manufacturer's instructions (PSL, Heidelberg, Germany). Secondary peroxidase-labeled anti-goat, anti-rabbit, and anti-mouse IgG were obtained from Sigma. Detection was performed with SuperSignal West Dura chemiluminescent substrate (Pierce) and a CCD camera (GDS 8000; UVP). Optical densities of Western signals were measured using the LabWorks 4.0 software (UVP). For quantification of PDE10 phosphorylation, pPDE10A and PDE10A in the same sample were measured, normalized to mean intensity of specific bands on a membrane to account for intensity variation of different blots, and expressed as pPDE/PDE ratio. Dynabeads (5 mg) with precipitated proteins were washed three times with PBS and resuspended in PKA buffer (10 μm cAMP, 0.5 mm ATP, 3 mm MgCl2, 0.5 mg/ml BSA, 2 mm dl-dithiothreitol, 50 mm triethanolamine/hydrochloride, pH 7.4) containing 1 μm MP-10 and PhosSTOP. In the negative control, cAMP and ATP were omitted. Incubation was for 10 min at 37 °C. Dynabeads were washed and proteins were eluted with 100 μl of SDS sample buffer. Volumes corresponding to 0.75 mg of Dynabeads were applied per lane of a 9% SDS gel. For gel filtration chromatography, the striata of 5 male mice were homogenized in Nonidet P-40 buffer (see above) and centrifuged for 30 min at 20,000 × g (4 °C). The resulting supernatant was applied to a HiLoad 26/60 Superdex 200pg column. The column was run with 0.68 column volumes of the Nonidet P-40 buffer containing 25 mm Tris, pH 7.4. Separation of proteins was followed by online detection of absorption at 280 nm. Fractions of 1.25 ml were collected and protein containing fractions were screened for PDE activity in the absence and presence of MP-10 (0.1 μm). For calibration, thyroglobulin (660 kDa), immunoglobulin G (150 kDa), bovine serum albumin (67 kDa), and ovalbumin (43 kDa) were separated on the same column and molecular weights were calculated using semi-logarithmic regression. Statistical testing was performed in Excel using Student's t test or Holm-Bonferroni's test (multiple comparisons of PDE activities with PDE inhibitors). For multiple comparisons of PDE10A phosphorylation, data were log transformed, homoscedasticity was checked by Levene's test, and data were further analyzed by one-way analysis of variance followed by Bonferroni's multiple comparisons post hoc tests using IBM SPSS 22. Differences are reported as significant at p < 0.05. Data are expressed as mean ± S.E. (error bars). Phosphodiesterases are critical regulators of cyclic nucleotide signaling in brain. In striatum, multiple PDEs with different substrate specificities and subcellular localization are expressed (11.Nishi A. Kuroiwa M. Miller D.B. O'Callaghan J.P. Bateup H.S. Shuto T. Sotogaku N. Fukuda T. Heintz N. Greengard P. Snyder G.L. Distinct roles of PDE4 and PDE10A in the regulation of cAMP/PKA signaling in the striatum.J. Neurosci. 2008; 28: 10460-10471Crossref PubMed Scopus (227) Google Scholar, 12.Sano H. Nagai Y. Miyakawa T. Shigemoto R. Yokoi M. Increased social interaction in mice deficient of the striatal medium spiny neuron-specific phosphodiesterase 10A2.J. Neurochem. 2008; 105: 546-556Crossref PubMed Scopus (84) Google Scholar, 22.Nishi A. Snyder G.L. Advanced research on dopamine signaling to develop drugs for the treatment of mental disorders: biochemical and behavioral profiles of phosphodiesterase inhibition in dopaminergic neurotransmission.J. Pharmacol. Sci. 2010; 114: 6-16Crossref PubMed Scopus (61) Google Scholar, 23.Russwurm C. Zoidl G. Koesling D. Russwurm M. Dual acylation of PDE2A splice variant 3: targeting to synaptic membranes.J. Biol. Chem. 2009; 284: 25782-25790Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 24.Lin D.T. Fretier P. Jiang C. Vincent S.R. Nitric oxide signaling via cGMP-stimulated phosphodiesterase in striatal neurons.Synapse. 2010; 64: 460-466Crossref PubMed Scopus (37) Google Scholar). Here, we set out to determine the relative contribution of the dual substrate PDE10A for cyclic nucleotide turnover in striatum. To this end, we measured cAMP and cGMP hydrolytic activity in striatal homogenates from mouse in the absence and presence of various PDE inhibitors at substrate concentrations of 0.1 μm cAMP (Fig. 1A) or 1 μm cGMP (Fig. 1B). Inhibition of PDEs 3, 4, 5, 9, or 11 did not affect cAMP or cGMP hydrolysis. Inhibition of PDE1 (50 μm vinpocetine) or PDE2 (0.2 μm Bay 60-7550) reduced the overall cAMP degradation by ∼10% each. The greatest effect on cAMP hydrolysis was observed in the presence of two different PDE10 inhibitors: papaverine (10 μm) inhibited ∼70%, and the more specific MP-10 (0.01 μm) inhibited ∼60% of cAMP hydrolysis (Fig. 1A). In the following, papaverine was used in some experiments because of the limited availability of MP-10. For cGMP turnover, PDEs 1 and 2 are most important as inhibition of these PDEs resulted in a ∼35 and ∼20% reduction of cGMP degradation, respectively. Although PDE10A is considered a dual specific PDE, its inhibition by papaverine only resulted in a slight reduction of cGMP-degrading activity in striatum (Fig. 1B). Thus, we conclude that PDE10A acts as the major cAMP PDE in striatum at the substrate concentration tested and controls most of the cAMP/PKA signal transduction in medium spiny neurons. Next we analyzed subcellular localization of PDE10A in mouse striatum. To assess compartmental localization, mice striata were separated into a cytosolic and a membrane fraction. Adequate fractionation was ensured by analysis of cytosolic and membrane marker proteins GAPDH and PSD95, respectively. As shown by Western immunodetection, PDE10A was enriched in the membrane fraction approximately ∼3-fold compared with the cytosolic fraction. These results are in accordance with several previous reports (10.Xie Z. Adamowicz W.O. Eldred W.D. Jakowski A.B. Kleiman R.J. Morton D.G. Stephenson D.T. Strick C.A. Williams R.D. Menniti F.S. Cellular and subcellular localization of PDE10A, a striatum-enriched phosphodiesterase.Neuroscience. 2006; 139: 597-607Crossref PubMed Scopus (179) Google Scholar, 25.Kotera J. Sasaki T. Kobayashi T. Fujishige K. Yamashita Y. Omori K. Subcellular localization of cyclic nucleotide phosphodiesterase type 10A variants, and alteration of the localization by cAMP-dependent protein kinase-dependent phosphorylation.J. Biol. Chem. 2004; 279: 4366-4375Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). In addition, we prepared synaptosomes. As shown in Fig. 1C, PDE10A distribution in the synaptosomal cytosol and membranes mirrored the distribution in total striatum. We therefore conclude that PDE10A is present in striatal synapses. PDE10A has been shown to be phosphorylated at Thr-16 by PKA in vitro and in heterologous expression systems (26.Kotera J. Fujishige K. Yuasa K. Omori K. Characterization and phosphorylation of PDE10A2, a novel alternative splice variant of human phosphodiesterase that hydrolyzes cAMP and cGMP.Biochem. Biophys. Res. Commun. 1999; 261: 551-557Crossref PubMed Scopus (72) Google Scholar, 27.Charych E.I. Jiang L.-X. Lo F. Sullivan K. Brandon N.J. Interplay of palmitoylation and phosphorylation in the trafficking and localization of phosphodiesterase 10A: implications for the treatment of schizophrenia.J. Neurosci. 2010; 30: 9027-9037Crossref PubMed Scopus (83) Google Scholar). However, evidence for phosphorylation to occur in vivo is sparse. To examine PDE10A2 phosphorylation in mouse striatum, we generated rabbit polyclonal antibodies that specifically recognize phosphorylated PDE10A2. This antibody reacted strongly with heterologously expressed PDE10A2 only after in vitro phosphorylation by PKA catalytic subunit (Fig. 2). In vivo phosphorylation of PDE10A2 was analyzed in mouse striatal slices. In lysate of untreated slices, phosphorylation of PDE10A2 was undetectable (Figs. 3A and 4, A and B, first lane). However, treatment with the adenylyl cyclase stimulator forskolin or the PDE10 inhibitors papaverine or MP-10 to raise intracellular cAMP and consecutively stimulate PKA led to a robust approximately 3–4-fold increase in phosphorylation (Figs. 3A and 4, A and B, respectively). This effect was enhanced by the simultaneous application of the PP2A/PP1 inhibitor okadaic acid (1 μm, Fig. 4A, third lane) 3The bands in the second and third lanes display similar density on the representative blot shown, which has been selected because it is representative for the other statistically significant differences. but not by cyclosporin A, a protein phosphatase 2B (calcineurin) inhibitor (Fig. 4B, third lane).FIGURE 4PDE10A phosphorylation is reversed by PP2A/PP1 inhibition. A and B, mouse striatal slices were preincubated with PDE10 inhibitors and (A) PP2A/PP1 or (B) PP2B phosphatase inhibitors a" @default.
• W2012249786 created "2016-06-24" @default.
• W2012249786 creator A5008681538 @default.
• W2012249786 creator A5048185763 @default.
• W2012249786 creator A5061884941 @default.
• W2012249786 date "2015-05-01" @default.
• W2012249786 modified "2023-09-27" @default.
• W2012249786 title "Phosphodiesterase 10A Is Tethered to a Synaptic Signaling Complex in Striatum" @default.
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