cAMP-regulated Phosphoprotein Research Articles

Abstract P3-06-10: t-Darpp enhances protein kinase A signaling through direct effects on PKA holoenzyme and indirectly influences cell metabolism, proliferation and apoptosis

Abstract Trastuzumab has dramatically improved the adjuvant therapy of Her2-positive breast cancer. However, acquired resistance to trastuzumab frequently occurs. Breast cancer cells in vitro selected for trastuzumab resistance exhibit over-expression of t-Darpp, a truncated form of the 32 kDa dopamine and cAMP-regulated phosphoprotein (Darpp-32). t-Darpp over-expression has previously been demonstrated to confer trastuzumab resistance and to result in increased protein kinase A (PKA) activity and signaling. Phosphorylation of Darpp-32 at threonine 75 has been shown to promote inhibition (not activation) of PKA by a yet unknown mechanism. T75 phosphorylation on t-Darpp is required for its trastuzumab resistance activity but the role of T75 phosphorylation in PKA activation by t-Darpp is not known and the molecular mechanism by which t-Darpp enhances PKA activity has not been reported. We used cells stably over-expressing either wild type t-Darpp or a T75A mutant of t-Darpp. Using proximity ligation assays, we found that t-Darpp binds directly to the RI regulatory subunit of PKA leading to 75% less RI subunit bound to the catalytic subunit of PKA (PKAc), compared to parental cells. In contrast, the t-Darpp-T75A mutant does not bind RI and those cells have only 34% less RI bound to PKAc compared to parental. These results suggest a direct effect of t-Darpp on PKA activation by binding the regulatory subunit RI and thereby preventing RI from inhibiting PKAc. The T75 phosphorylation site appears to be required for this activity. Using enzymatic and caspase activity assays, we also show that cells over-expressing t-Darpp, but not t-Darpp-T75A, have increased proliferation, up to 4-fold more cellular ATP, and diminished apoptotic response to etoposide, relative to parental cell controls. So far, this data suggests a dual mode of t-Darpp action: modulation of cell metabolism (possibly via effects on ATP production) and direct effects on PKA holoenzyme. Ultimately, pro-apoptotic response is diminished. Further studies will determine whether PKA activation by t-Darpp is responsible for, or separately influenced by, the metabolic effects of t-Darpp. Citation Format: Lenz G, Theile D, Kane SE. t-Darpp enhances protein kinase A signaling through direct effects on PKA holoenzyme and indirectly influences cell metabolism, proliferation and apoptosis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-06-10.

DARPP-32: from neurotransmission to cancer.

Dopamine and cAMP-regulated phosphoprotein Mr 32,000 (DARPP-32), also known as phosphoprotein phosphatase-1 regulatory subunit 1B (PPP1R1B), was initially discovered as a substrate of dopamine-activated protein kinase A (PKA) in the neostriatum in the brain. While phosphorylation at Thr-34 by PKA converts DARPP-32 into a potent inhibitor of protein phosphatase 1 (PP1), phosphorylation at Thr-75 transforms DARPP-32 into an inhibitor of PKA. Through regulation of DARPP-32 phosphorylation and modulation of protein phosphatase and kinase activities, DARPP-32 plays a critical role in mediating the biochemical, electrophysiological, and behavioral effects controlled by dopamine and other neurotransmitters in response to drugs of abuse and psychostimulants. Altered expression of DARPP-32 and its truncated isoform (t-DARPP), specifically in the prefrontal cortex, has been associated with schizophrenia and bipolar disorder. Moreover, cleavage of DARPP-32 by calpain has been implicated in Alzheimer's disease. Amplification of the genomic locus of DARPP-32 at 17q12 has been described in several cancers. DARPP-32 and t-DARPP are frequently overexpressed at the mRNA and protein levels in adenocarcinomas of the breast, prostate, colon, and stomach. Several studies demonstrated the pro-survival, pro-invasion, and pro-angiogenic functions of DARPP-32 in cancer. Overexpression of DARPP-32 and t-DARPP also promotes chemotherapeutic drug resistance and cell proliferation in gastric and breast cancers through regulation of pro-oncogenic signal transduction pathways. The expansion of DARPP-32 research from neurotransmission to cancer underscores the broad scope and implication of this protein in disparate human diseases.

Unraveling the Mechanism of Dyskinesia One Transcription Factor at a Time

L-DOPA–induced dyskinesia (LID), or abnormal involuntary movements, is a common side effect of L-DOPA therapy in patients with Parkinson’s disease. When L-DOPA is initially administered, it effectively reverses akinesia caused by the loss of dopamine. However, long-term L-DOPA administration produces a hyperkinetic effect, triggering excessive movements in human patients and in animal models. The mechanisms of LID have been under intense investigation for decades, and numerous important discoveries in the past several years established the key mechanisms of LID development. Five dopamine receptor subtypes in the striatal neurons mediate the action of dopamine produced from L-DOPA. The main subtypes, D1 and D2, are expressed on the striatonigral and striatopallidal medium spiny neurons (MSNs), respectively, with a high degree of segregation. The relative contribution of the D1-dependent and D2-dependent signaling to LID has been hotly debated. However, findings demonstrating that genetic ablation of the D1, but not the D2, receptor abrogates LID (1) strongly suggest that the aberrant signaling via the D1 receptor is the main contributor. The striking molecular feature of the dopamine-depleted striatum is the hyperresponsiveness of multiple signaling pathways to acute dopaminergic stimulation. The D1 receptor in MSNs couples to the adenylyl cyclase–stimulating Golf, and in the dopamine-depleted striatum, the cyclic adenosine monophosphate (cAMP) response to L-DOPA is augmented, resulting in the increased activity of protein kinase A (PKA) and enhanced phosphorylation of PKA substrates. One of the substrates, the dopamine and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), upon phosphorylation by PKA at Thr34, acts as an inhibitor of protein phosphatase 1, further enhancing the activity of the cAMP-PKA cascade. Sensitized DARPP-32 contributes to LID because its genetic ablation or elimination of the PKA-phosphorylated residue ameliorates LID (2,3). In addition, DARPP-32 inactivation significantly attenuates the extracellular signal-regulated kinase (ERK) superactivity in the dopamine-depleted striatum (2), suggesting that the elevated cAMP signaling is an important mediator of the ERK superactivity. The ERK pathway shows very strong dopamine depletion– induced supersensitivity. The dopamine-induced hyperactivation of the ERK response is confined to the striatonigral MSNs, or direct pathway MSNs (dMSNs) (1,3), and genetic ablation of the D1 receptor prevents the supersensitive ERK response (1), reinforcing the notion of the prime role of the D1 receptor signaling in LID. The neuron-specific Ras–guanine nucleotidereleasing factor 1 (Ras-GRF1), the upstream activator of Ras, implicated in LID could serve as the mechanistic connection between the D1 receptor signaling and activation of the ERK

Basal Ganglia Damage in Experimental Subarachnoid Hemorrhage.

Research suggests that early brain injury following subarachnoid hemorrhage (SAH) is a primary therapeutic target, and early SAH-induced basal ganglia injury is not well studied. The present study examined basal ganglia injury in a rat model of SAH. Adult male Sprague-Dawley rats (n = 78) weighing 275-300 g underwent endovascular perforation to mimic aneurysmal SAH. Sham rats (n = 12) underwent the same procedure but without perforation. Magnetic resonance imaging (T2 MRI) was performed at 24 h after SAH to measure ventricle volumes and brain T2 lesion. Hydrocephalus in SAH rats was defined as a ventricular volume greater than three standard deviations above that in shams. Western blotting and immunochemistry were utilized to assess basal ganglia damage. Sixty rats survived the SAH and 40 % of those animals had T2 lesions in the basal ganglia. Twenty-six SAH rats had hydrocephalus. Rats with hydrocephalus had higher incidence of basal ganglia lesion (69 vs. 18 % in rats without hydrocephalus; p < 0.01). Basal ganglia neuronal injury was also determined by examining the levels of dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa (DARPP-32). We found that rats with hydrocephalus had more severe basal ganglia injury with greater DARPP-32 depletion (DARPP-32/beta-actin: 0.38 ± 0.32 vs. 0.86 ± 0.45 in rats without hydrocephalus and 1.10 ± 0.28 in sham, p < 0.05). In conclusion, SAH resulted in severe basal ganglia damage, which is associated with hydrocephalus development.

The use of matrix coating assisted by an electric field (MCAEF) to enhance mass spectrometric imaging of human prostate cancer biomarkers.

In this work, we combined a newly developed matrix coating technique - matrix coating assisted by an electric field (MCAEF) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to enhance the imaging of peptides and proteins in tissue specimens of human prostate cancer. MCAEF increased the signal-to-noise ratios of the detected proteins by a factor of 2 to 5, and 232 signals were detected within the m/z 3500-37500 mass range on a time-of-flight mass spectrometer and with the sinapinic acid MALDI matrix. Among these species, three proteins (S100-A9, S100-A10, and S100-A12) were only observed in the cancerous cell region and 14 proteins, including a fragment of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 2, a fragment of cAMP-regulated phosphoprotein 19, 3 apolipoproteins (C-I, A-I, and A-II), 2 S100 proteins (A6 and A8), β-microseminoprotein, tumor protein D52, α-1-acid glycoprotein 1, heat shock protein β-1, prostate-specific antigen, and 2 unidentified large peptides at m/z 5002.2 and 6704.2, showed significantly differential distributions at the p < 0.05 (t-test) level between the cancerous and the noncancerous regions of the tissue. Among these 17 species, the distributions of apolipoprotein C-I, S100-A6, and S100-A8 were verified by immunohistological staining. In summary, this study resulted in the imaging of the largest group of proteins in prostate cancer tissues by MALDI-MS reported thus far, and is the first to show a correlation between S100 proteins and prostate cancer in a MS imaging study. The successful imaging of the three proteins only found in the cancerous tissues, as well as those showing differential expressions demonstrated the potential of MCAEF-MALDI/MS for the in situ detection of potential cancer biomarkers. Copyright © 2015 John Wiley & Sons, Ltd.

Motor Skill Learning Is Associated with Phase-Dependent Modifications in the Striatal cAMP/PKA/DARPP-32 Signaling Pathway in Rodents.

Abundant evidence points to a key role of dopamine in motor skill learning, although the underlying cellular and molecular mechanisms are still poorly understood. Here, we used a skilled-reaching paradigm to first examine changes in the expression of the plasticity-related gene Arc to map activity in cortico-striatal circuitry during different phases of motor skill learning in young animals. In the early phase, Arc mRNA was significantly induced in the medial prefrontal cortex (mPFC), cingulate cortex, primary motor cortex, and striatum. In the late phase, expression of Arc did not change in most regions, except in the mPFC and dorsal striatum. In the second series of experiments, we studied the learning-induced changes in the phosphorylation state of dopamine and cAMP-regulated phosphoprotein, 32k Da (DARPP-32). Western blot analysis of the phosphorylation state of DARPP-32 and its downstream target cAMP response element-binding protein (CREB) in the striatum revealed that the early, but not late, phase of motor skill learning was associated with increased levels of phospho-Thr34-DARPP-32 and phospho-Ser133-CREB. Finally, we used the DARPP-32 knock-in mice with a point mutation in the Thr34 regulatory site (i.e., protein kinase A site) to test the significance of this pathway in motor skill learning. In accordance with our hypothesis, inhibition of DARPP-32 activity at the Thr34 regulatory site strongly attenuated the motor learning rate and skilled reaching performance of mice. These findings suggest that the cAMP/PKA/DARPP-32 signaling pathway is critically involved in the acquisition of novel motor skills, and also demonstrate a dynamic shift in the contribution of cortico-striatal circuitry during different phases of motor skill learning.

Deficits in behavioral sensitization and dopaminergic responses to methamphetamine in adenylyl cyclase 1/8-deficient mice.

The cAMP/protein kinase A pathway regulates methamphetamine (METH)‐induced neuroplasticity underlying behavioral sensitization. We hypothesize that adenylyl cyclases (AC) 1/8 mediate these neuroplastic events and associated striatal dopamine regulation. Locomotor responses to METH (1 and 5 mg/kg) and striatal dopamine function were evaluated in mice lacking AC 1/8 (DKO) and wild‐type (WT) mice. Only 5 mg/kg METH induced an acute locomotor response in DKO mice, which was significantly attenuated versus WT controls. DKO mice showed a marked attenuation in the development and expression of METH‐induced behavioral sensitization across doses relative to WT controls. While basal and acute METH (5 mg/kg)‐evoked accumbal dialysate dopamine levels were similar between genotypes, saline‐treated DKO mice showed elevated tissue content of dopamine and homovanillic acid in the dorsal striatum (DS), reflecting dysregulated dopamine homeostasis and/or metabolism. Significant reductions in DS dopamine levels were observed in METH‐sensitized DKO mice compared to saline‐treated controls, an effect not observed in WT mice. Notably, saline‐treated DKO mice had significantly increased phosphorylated Dopamine‐ and cAMP‐regulated phosphoprotein levels, which were not further augmented following METH sensitization, as observed in WT mice. These data indicate that AC 1/8 are critical to mechanisms subserving drug‐induced behavioral sensitization and mediate nigrostriatal pathway METH sensitivity. Calcium/calmodulin‐stimulated adenylyl cyclase (AC) isoforms 1 and 8 were studied for their involvement in the adaptive neurobehavioral responses to methamphetamine. AC 1/8 double knockout (DKO) mice showed heightened basal locomotor activity and dorsal striatal dopamine responsivity. Conversely, methamphetamine‐induced locomotor activity was attenuated in DKO mice, accompanied by reductions in dopamine and HVA content and impaired DARPP‐32 activation. These findings indicate AC 1/8 signaling regulates the sensitivity of the nigrostriatal pathway subserving stimulant and neuroadaptive sensitizing effects of methamphetamine. 3‐MT, 3‐methoxytyramine; Ca2+, calcium; CaM, calmodulin; cdk5; cyclin‐dependent kinase 5; DA, dopamine; DARPP‐32, dopamine‐ and cAMP‐regulated phosphoprotein; D1R, dopamine D1 receptor; HVA, homovanillic acid; PKA, protein kinase A.

Micro-transplantation of ganglionic eminence cells reduces Huntington-associated phenotypes in rats

Objective To compare the therapeutic effects of microtransplantation method and traditional transplantation method for transplanting ganglionic eminence cell into HD rats. Methods Seventy-five adult female Sprague-Dawley rats were randomly divided into four groups, control group (ctrl; n=15), sham transplantation group (ST; n=20), traditional transplantation group (TT; n=20), and microtransplantation group (MT; n=20). Ganglionic eminence cells or phosphate-buffered saline were transplanted into unilateral striatum of quinic acid lesion rats with micro-transplantation instruments (with an outer diameter of 50 μm) or traditional transplantation instruments (with an outer diameter of 470 μm). Apomorphine-induced rotation test and adjusting step test were assessed after QA-induced lesion and 2, 4, 6, 8, 10 and 12 weeks after transplantation. The expression of neuronal nuclei (NeuN), dopamine, cAMP-regulated phosphoprotein of molecular weight 32 kDa (DARPP-32), and glial fibrillary acidic protein (GFAP) were analyzed at 12 weeks after transplantation. Results Compared with traditional transplantation group, microtransplantation group performed well in stepping test. DARPP-32 positives cell in MT group(13 194±976) had a significantly larger number compared with TT group(9 690±651) (P=0.020). The TT group(11 352± 1 421) had a significantly larger number of GFAP-positive cells compared with MT groups (6 558±694)(P=0.002). The survival rate of the MT group(85%) during the 12 weeks after transplantation was significantly higher than that of the TT groups(55%)(P=0.012). Conclusion Micro-transplantation approach can minimize the trauma associated with transplantation and also provide accurate targets in the host brain. It may be an alternative transplantation strategy for Huntington's disease. Key words: Ganglionic eminence cell; Micro-transplantation; Rat model of Huntington's disease; Cell survival; Functional recovery

Calpain-mediated cleavage of DARPP-32 in Alzheimer's disease.

Toxicity induced by aberrant protein aggregates in Alzheimer’s disease (AD) causes synaptic disconnection and concomitant progressive neurodegeneration that eventually impair cognitive function. cAMP-response element-binding protein (CREB) is a transcription factor involved in the molecular switch that converts short-term to long-term memory. Although disturbances in CREB function have been suggested to cause memory deficits in both AD and AD animal models, the mechanism of CREB dysfunction is still unclear. Here, we show that the dopamine- and cAMP-regulated phosphoprotein 32 kDa (DARPP-32), a key inhibitor of protein phosphate-1 (PP-1) that regulates CREB phosphorylation, is cleaved by activated calpain in both AD brains and neuronal cells treated with amyloid-β or okadaic acid, a protein phosphatase-2A inhibitor that induces tau hyperphosphorylation and neuronal death. We found that DARPP-32 is mainly cleaved at Thr153 by calpain and that this cleavage of DARPP-32 reduces CREB phosphorylation via loss of its inhibitory function on PP1. Our results suggest a novel mechanism of DARPP-32–CREB signalling dysregulation in AD.

Activation of Protein Kinases and Phosphatases Coupled to Glutamate Receptors Regulates the Phosphorylation State of DARPP32 at Threonine 75 After Repeated Exposure to Cocaine in the Rat Dorsal Striatum in a Ca2+-Dependent Manner.

Background:Phosphorylation state of dopamine- and cAMP-regulated phosphoprotein, molecular weight 32kDa (DARPP32) is crucial to understand drug-mediated synaptic plasticity. In this study, mechanisms underlying repeated cocaine-stimulated phosphorylation of DARPP32 at threonine 75 (pDARPP32-Thr75) were determined by investigating the hypothesis that activation of protein kinases and phosphatases coupled to glutamate signaling is necessary for the regulation of pDARPP32-Thr75 after repeated cocaine administration.Methods:Intracaudate drug infusions into the rat dorsal striatum followed by Western immunoblot analysis were mainly performed to test this hypothesis.Results:The results demonstrated that 7 repeated daily intraperitoneal injections of cocaine (20mg/kg) upregulated the expression of pDARPP32-Thr75. Increases in the cytosolic Ca2+ concentrations followed by Ca2+-dependent protein kinase activation through stimulation of Ca2+ channels in striatal neurons were necessary for the phosphorylation. Activation of protein phosphatases further regulated the phosphorylation state by deactivating pDARPP32-Thr75 and upstream protein kinases.Conclusion:These findings suggest that activation of protein kinases and phosphatases coupled to glutamate receptors controls the phosphorylation state of DARPP32-Thr75 after repeated exposure to cocaine in the dorsal striatum in a Ca2+-dependent manner.

Selective Effects of PDE10A Inhibitors on Striatopallidal Neurons Require Phosphatase Inhibition by DARPP-32

Type 10A phosphodiesterase (PDE10A) is highly expressed in the striatum, in striatonigral and striatopallidal medium-sized spiny neurons (MSNs), which express D1 and D2 dopamine receptors, respectively. PDE10A inhibitors have pharmacological and behavioral effects suggesting an antipsychotic profile, but the cellular bases of these effects are unclear. We analyzed the effects of PDE10A inhibition in vivo by immunohistochemistry, and imaged cAMP, cAMP-dependent protein kinase A (PKA), and cGMP signals with biosensors in mouse brain slices. PDE10A inhibition in mouse striatal slices produced a steady-state increase in intracellular cAMP concentration in D1 and D2 MSNs, demonstrating that PDE10A regulates basal cAMP levels. Surprisingly, the PKA-dependent AKAR3 phosphorylation signal was strong in D2 MSNs, whereas D1 MSNs remained unresponsive. This effect was also observed in adult mice in vivo since PDE10A inhibition increased phospho-histone H3 immunoreactivity selectively in D2 MSNs in the dorsomedial striatum. The PKA-dependent effects in D2 MSNs were prevented in brain slices and in vivo by mutation of the PKA-regulated phosphorylation site of 32 kDa dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is required for protein phosphatase-1 inhibition. These data highlight differences in the integration of the cAMP signal in D1 and D2 MSNs, resulting from stronger inhibition of protein phosphatase-1 by DARPP-32 in D2 MSNs than in D1 MSNs. This study shows that PDE10A inhibitors share with antipsychotic medications the property of activating preferentially PKA-dependent signaling in D2 MSNs.

Role of Dopamine Type 1 Receptors and Dopamine- and cAMP-Regulated Phosphoprotein Mr 32 kDa in Δ9-Tetrahydrocannabinol-Mediated Induction of ΔFosB in the Mouse Forebrain.

Δ(9)-Tetrahydrocannabinol (THC), the main psychoactive component of marijuana, produces motor and motivational effects via interactions with the dopaminergic system in the caudate-putamen and nucleus accumbens. However, the molecular events that underlie these interactions after THC treatment are not well understood. Our study shows that pretreatment with dopamine D1 receptor (D1R) antagonists before repeated administration of THC attenuated induction of Δ FBJ murine osteosarcoma viral oncogene homolog B (ΔFosB) in the nucleus accumbens, caudate-putamen, amygdala, and prefrontal cortex. Anatomical studies showed that repeated THC administration induced ΔFosB in D1R-containing striatal neurons. Dopamine signaling in the striatum involves phosphorylation-specific effects of the dopamine- and cAMP-regulated phosphoprotein Mr 32 kDa (DARPP-32), which regulates protein kinase A signaling. Genetic deletion of DARPP-32 attenuated ΔFosB expression measured after acute, but not repeated, THC administration in both the caudate-putamen and nucleus accumbens. THC was then acutely or repeatedly administered to wild-type (WT) and DARPP-32 knockout (KO) mice, and in vivo responses were measured. DARPP-32 KO mice exhibited enhanced acute THC-mediated hypolocomotion and developed greater tolerance to this response relative to the WT mice. Agonist-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding showed that cannabinoid-stimulated G-protein activity did not differ between DARPP-32 KO and WT mice treated with vehicle or repeated THC. These results indicate that D1Rs play a major role in THC-mediated ΔFosB induction in the forebrain, whereas the role of DARPP-32 in THC-mediated ΔFosB induction and modulation of motor activity appears to be more complex.

Epigenetic modulation of brain gene networks for cocaine and alcohol abuse.

Cocaine and alcohol are two substances of abuse that prominently affect the central nervous system (CNS). Repeated exposure to cocaine and alcohol leads to longstanding changes in gene expression, and subsequent functional CNS plasticity, throughout multiple brain regions. Epigenetic modifications of histones are one proposed mechanism guiding these enduring changes to the transcriptome. Characterizing the large number of available biological relationships as network models can reveal unexpected biochemical relationships. Clustering analysis of variation from whole-genome sequencing of gene expression (RNA-Seq) and histone H3 lysine 4 trimethylation (H3K4me3) events (ChIP-Seq) revealed the underlying structure of the transcriptional and epigenomic landscape within hippocampal postmortem brain tissue of drug abusers and control cases. Distinct sets of interrelated networks for cocaine and alcohol abuse were determined for each abusive substance. The network approach identified subsets of functionally related genes that are regulated in agreement with H3K4me3 changes, suggesting cause and effect relationships between this epigenetic mark and gene expression. Gene expression networks consisted of recognized substrates for addiction, such as the dopamine- and cAMP-regulated neuronal phosphoprotein PPP1R1B/DARPP-32 and the vesicular glutamate transporter SLC17A7/VGLUT1 as well as potentially novel molecular targets for substance abuse. Through a systems biology based approach our results illustrate the utility of integrating epigenetic and transcript expression to establish relevant biological networks in the human brain for addiction. Future work with laboratory models may clarify the functional relevance of these gene networks for cocaine and alcohol, and provide a framework for the development of medications for the treatment of addiction.

Correlating Cerebral (18)FDG PET-CT Patterns with Histological Analysis During Early Brain Injury in a Rat Subarachnoid Hemorrhage Model.

Early brain injury (EBI) plays a significant role in poor outcomes for subarachnoid hemorrhage (SAH) patients. Further investigations are required to characterize the cellular metabolic and related histological changes that may contribute to EBI following SAH. We investigated the image patterns of 18-fluorodeoxyglucose positron emission tomography-computed tomography ((18)FDG PET-CT) during EBI and correlated histopathological changes utilizing a rat SAH model. SAH was induced in six adult male Sprague-Dawley rats by endovascular perforation, and animals were randomly assigned to receive (18)FDG PET-CT imaging at either 3 or 12h post-procedure. Mean (18)FDG standard uptake value (SUV) of the brain was calculated. Animals were euthanized 48h post-procedure, and brain samples were used for heme oxygenase-1 (HO-1) and dopamine- and cAMP-regulated phosphoprotein (DARPP-32) Mr 32kDa immunohistochemistry. Rats within the SAH group had higher mean whole brain (18)FDG SUV (2.349 ± 0.376g/ml in the 3-h group and 2.453 ± 0.495g/ml in the 12-h group) compared to that of sham (n = 3; mean SUV = 2.030 ± 0.247g/ml; P < 0.05) or control groups (n = 3; mean SUV = 1.800 ± 0.484g/ml; P < 0.05). Whole brain (18)FDG SUV did not vary significantly between rats imaged at 3h vs. those imaged at 12h post-SAH (P > 0.05). Regions of decreasing SUV in SAH rats correlated with neuronal death and increased expression of HO-1. Higher (18)FDG PET SUV was evident in rats post-SAH compared to sham and control groups. Regions of decreasing SUV in SAH rats correlated with neuronal death and increased HO-1 expression as evaluated by histopathology.

409 Factors Relating to GI Bleeding Induced by Dual Antiplatelet Therapy (DAPT) in Japanese Patients With Percutaneous Coronary Intervention - Retrospective Cohort and Case Control Studies

Background: Splicing and expression of alternative exons is a tissue specific feature that affects the cancer genome. Notably, pre-mRNA-splicing factor SRp20 plays an important role in regulating alternative splicing. Expression of CD44 and its splicing variants have been shown to regulate carcinogenesis and invasion. Herein, we investigated factors that regulate splicing of CD44 and expression of its variant CD44E in gastric cancer. We identified a novel role for dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32), in regulating CD44 splicing and promoting gastric tumorigenesis. Methods:Pre-mRNA splicing was measured by utilizing a luciferase-based quantitative reporter system. Quantitative realtime RT-PCR and Western blot analysis were used to measure the mRNA and protein levels. Co-immunoprecipitation, invasion, and ubiquitination assays were performed to investigate the mechanism by which DARPP-32 regulates CD44 splicing . Results: Knockdown of endogenous DARPP-32 in MKN45 gastric cancer cells markedly reduced expression of CD44 V8-V10 (CD44E) splice variant. We found that overexpression of DARPP-32 in AGS gastric cancer cells, with low endogenous levels, significantly increased the splicing activity (P<0.001). Conversely, knocking down endogenous DARPP-32 significantly attenuated the splicing activity (P<0.001). Mechanistic investigations revealed that modulation of DARPP-32 was associated with changes in splicing factor SRp20 expression through a posttranslational mechanism that involves the binding of DARPP-32 and SRp20 in the same protein complex. Inhibition of alternative splicing with digitoxin followed by immunoprecipitation and immunoblotting indicated that DARPP-32 enhances SRp20 protein stability through regulation of ubiquitination. In contrast, the knockdown of endogenous DARPP-32 reversed these effects and indicated that DARPP-32 regulated the SRp20-dependent CD44E splicing. In a xenograft tumor mouse model, knocking down endogenous DARPP-32 markedly decreased tumor growth, and overexpression of SRp20 rescued tumor growth. Importantly, our clinical data demonstrated frequent co-overexpression and positive correlation of DARPP-32, SRp20 and CD44E in human gastric primary tumors, strongly supporting a role for DARPP-32 in regulating CD44E splicing in gastric cancer. Conclusion: These novel findings establish for the first time that DARPP-32-SRp20 axis plays a key role in regulating the expression of CD44E splice variant in gastric tumorigenesis. This novel DARPP-32-mediated splicingdependent mechanism provides a new paradigm in cancer invasion.

Delayed post-treatment with bone marrow-derived mesenchymal stem cells is neurorestorative of striatal medium-spiny projection neurons and improves motor function after neonatal rat hypoxia-ischemia.

Perinatal hypoxia-ischemia is a major cause of striatal injury and may lead to cerebral palsy. This study investigated whether delayed administration of bone marrow-derived mesenchymal stem cells (MSCs), at one week after neonatal rat hypoxia-ischemia, was neurorestorative of striatal medium-spiny projection neurons and improved motor function. The effect of a subcutaneous injection of a high-dose, or a low-dose, of MSCs was investigated in stereological studies. Postnatal day (PN) 7 pups were subjected to hypoxia-ischemia. At PN14, pups received treatment with either MSCs or diluent. A subset of high-dose pups, and their diluent control pups, were also injected intraperitoneally with bromodeoxyuridine (BrdU), every 24h, on PN15, PN16 and PN17. This permitted tracking of the migration and survival of neuroblasts originating from the subventricular zone into the adjacent injured striatum. Pups were euthanized on PN21 and the absolute number of striatal medium-spiny projection neurons was measured after immunostaining for DARPP-32 (dopamine- and cAMP-regulated phosphoprotein-32), double immunostaining for BrdU and DARPP-32, and after cresyl violet staining alone. The absolute number of striatal immunostained calretinin interneurons was also measured. There was a statistically significant increase in the absolute number of DARPP-32-positive, BrdU/DARPP-32-positive, and cresyl violet-stained striatal medium-spiny projection neurons, and fewer striatal calretinin interneurons, in the high-dose mesenchymal stem cell (MSC) group compared to their diluent counterparts. A high-dose of MSCs restored the absolute number of these neurons to normal uninjured levels, when compared with previous stereological data on the absolute number of cresyl violet-stained striatal medium-spiny projection neurons in the normal uninjured brain. For the low-dose experiment, in which cresyl violet-stained striatal medium-spiny neurons alone were measured, there was a lower statistically significant increase in their absolute number in the MSC group compared to their diluent controls. Investigation of behavior in another cohort of animals showed that delayed administration of a high-dose of bone marrow-derived MSCs, at one week after neonatal rat hypoxia-ischemia, improved motor function on the cylinder test. Thus, delayed therapy with a high- or low-dose of adult MSCs, at one week after injury, is effective in restoring the loss of striatal medium-spiny projection neurons after neonatal rat hypoxia-ischemia and a high-dose of MSCs improved motor function.

Gastric tumour-derived ANGPT2 regulation by DARPP-32 promotes angiogenesis

ObjectiveOverexpression of dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32), and its truncated isoform (t-DARPP) are associated with gastric tumorigenesis. Herein, we investigated the role of DARPP-32 proteins in regulating angiopoietin...

MicroRNA-320a sensitizes tamoxifen-resistant breast cancer cells to tamoxifen by targeting ARPP-19 and ERRγ.

Tamoxifen represents a major adjuvant therapy to those patients with estrogen receptor-alpha positive breast cancer. However, tamoxifen resistance occurs quite often, either de novo or acquired during treatment. To investigate the role of miR-320a in the development of resistance to tamoxifen, we established tamoxifen-resistant (TamR) models by continually exposing MCF-7 or T47D breast cancer cells to tamoxifen, and identified microRNA(miRNA)-320a as a down-regulated miRNA in tamoxifen resistant cells. Re-expression of miR-320a was sufficient to sensitize TamR cells to tamoxifen by targeting cAMP-regulated phosphoprotein (ARPP-19) and estrogen-related receptor gamma (ERRγ) as well as their downstream effectors, c-Myc and Cyclin D1. Furthermore, progesterone (P4) promoted the expression of miR-320a by repressing c-Myc expression, while estrogen (E2) exerted the opposite effect. These results suggest the potential therapeutic approach for tamoxifen-resistant breast cancer by restorating miR-320a expression or depleting ARPP-19/ERRγ expression.

Chronic baclofen desensitizes GABAB-mediated G-protein activation and stimulates phosphorylation of kinases in mesocorticolimbic rat brain

The GABAB receptor is a therapeutic target for CNS and neuropathic disorders; however, few preclinical studies have explored effects of chronic stimulation. This study evaluated acute and chronic baclofen treatments on GABAB-activated G-proteins and signaling protein phosphorylation as indicators of GABAB signaling capacity. Brain sections from rats acutely administered baclofen (5 mg/kg, i.p.) showed no significant differences from controls in GABAB-stimulated GTPγS binding in any brain region, but displayed significantly greater phosphorylation/activation of focal adhesion kinase (pFAKTyr397) in mesocorticolimbic regions (caudate putamen, cortex, hippocampus, thalamus) and elevated phosphorylated/activated glycogen synthase kinase 3-β (pGSK3βTyr216) in the prefrontal cortex, cerebral cortex, caudate putamen, nucleus accumbens, thalamus, septum, and globus pallidus. In rats administered chronic baclofen (5 mg/kg, t.i.d. for five days), GABAB-stimulated GTPγS binding was significantly diminished in the prefrontal cortex, septum, amygdala, and parabrachial nucleus compared to controls. This effect was specific to GABAB receptors: there was no effect of chronic baclofen treatment on adenosine A1-stimulated GTPγS binding in any region. Chronically-treated rats also exhibited increases in pFAKTyr397 and pGSK3βTyr216 compared to controls, and displayed wide-spread elevations in phosphorylated dopamine- and cAMP-regulated phosphoprotein-32 (pDARPP-32Thr34) compared to acutely-treated or control rats. We postulate that those neuroadaptive effects of GABAB stimulation mediated by G-proteins and their sequelae correlate with tolerance to several of baclofen's effects, whereas sustained signaling via kinase cascades points to cross-talk between GABAB receptors and alternative mechanisms that are resistant to desensitization. Both desensitized and sustained signaling pathways should be considered in the development of pharmacotherapies targeting the GABA system.

Impramine, fluoxetine and clozapine differently affected reactivity to positive and negative stimuli in a model of motivational anhedonia in rats

Anhedonia is a relevant symptom in depression and schizophrenia. Chronic stress exposure induces in rats escape deficit, disrupts the dopaminergic response to palatable food and the competence to acquire sucrose self-administration (SA), thus configuring a possible model of motivational anhedonia. Repeated lithium administration reverts stress effects and brings back to control values the breaking point (BP) score, a measure of reward motivation. In this study, we tested on this model two antidepressants, imipramine and fluoxetine, and two antipsychotics, haloperidol and clozapine. The dopaminergic response to sucrose consumption was studied in non food-deprived rats in terms of dopamine D1 receptor signaling in the nucleus accumbens shell (NAcS). More specifically, we studied the modifications in dopamine and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32) phosphorylation pattern following sucrose consumption. Fluoxetine reverted the escape deficit and showed no effects on dopaminergic response and sucrose SA. Imipramine reverted sucrose SA and dopamine response deficit in half of the rats and the escape deficit in all animals. Haloperidol did not affect stress-induced deficits. Clozapine-treated rats recovered the dopaminergic response to sucrose consumption and the competence to acquire sucrose SA, although they still showed the escape deficit, thus confirming that motivation toward reward may be dissociated from that to punishment escape. These results indicate that imipramine or fluoxetine are not endowed with a rapid onset antianhedonic effect. On the other hand, clozapine treatment showed a motivational antianhedonic activity similar to that observed after lithium treatment.