t-Darpp overexpression in HER2-positive breast cancer confers a survival advantage in lapatinib.

Drug resistance is a major barrier to successful cancer treatment. For patients with HER2-positive breast cancer who initially respond to therapy, the majority develop resistance within one year of treatment. Patient outcomes could improve significantly if we can find and exploit common mechanisms of acquired resistance to different targeted therapies. Overexpression of t-Darpp, a truncated form of the dual kinase/phosphatase inhibitor Darpp-32, has been linked to acquired resistance to trastuzumab, a front-line therapy for HER2-positive breast cancer. Darpp-32 reverses t-Darpp's effect on trastuzumab resistance. In this study, we examined whether t-Darpp could be involved in resistance to lapatinib, another HER2-targeted therapeutic. Lapatinib-resistant SKBR3 cells (SK/LapR) showed a marked change in the Darpp-32:t-Darpp ratio toward a predominance of t-Darpp. Overexpression of t-Darpp alone was not sufficient to confer lapatinib resistance, but cells that overexpress t-Darpp partially mimicked the molecular resistance phenotype observed in SK/LapR cells exposed to lapatinib. SK/LapR cells failed to down-regulate Survivin and failed to induce BIM accumulation in response to lapatinib; cells overexpressing t-Darpp exhibited only the failed BIM accumulation. t-Darpp knock-down reversed this phenotype. Using a fluorescence-based co-culture system, we found that cells overexpressing t-Darpp formed colonies in lapatinib within 3-4 weeks, whereas parental cells in the same co-culture did not. Overall, t-Darpp appears to mediate a survival advantage in lapatinib, possibly linked to failed lapatinib-induced BIM accumulation. t-Darpp might also be relevant to acquired resistance to other cancer drugs that rely on BIM accumulation to induce apoptosis.

Elucidating the role of the A2A adenosine receptor in neurodegeneration using neurons derived from Huntington's disease iPSCs.

Huntington's disease (HD) is an autosomal-dominant degenerative disease caused by a cytosine-adenine-guanine trinucleotide expansion in the Huntingtin (htt) gene. The most vulnerable brain areas to mutant HTT-evoked toxicity are the striatum and cortex. In spite of the extensive efforts that have been devoted to the characterization of HD pathogenesis, no disease-modifying therapy for HD is currently available. The A2A adenosine receptor (A2AR) is widely distributed in the brain, with the highest level observed in the striatum. We previously reported that stimulation of the A2AR triggers an anti-apoptotic effect in a rat neuron-like cell line (PC12). Using a transgenic mouse model (R6/2) of HD, we demonstrated that A2AR-selective agonists effectively ameliorate several major symptoms of HD. In the present study, we show that human iPSCs can be successfully induced to differentiate into DARPP32-positive, GABAergic neurons which express the A2AR in a similar manner to striatal medium spiny neurons. When compared with those derived from control subjects (CON-iPSCs), these HD-iPSC-derived neurons exhibited a higher DNA damage response, based on the observed expression of γH2AX and elevated oxidative stress. This is a critical observation, because oxidative damage and abnormal DNA damage/repair have been reported in HD patients. Most importantly, stimulation of the A2AR using selective agonists reduced DNA damage and oxidative stress-induced apoptosis in HD-iPSC-derived neurons through a cAMP/PKA-dependent pathway. These findings support our hypothesis that human neurons derived from diseased iPSCs might serve as an important platform to investigate the beneficial effects and underlying mechanisms of A2AR drugs.

Regulation of CXCR4-mediated invasion by DARPP-32 in gastric cancer cells.

Although Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) is overexpressed in two-thirds of gastric cancers, its impact on molecular functions has not been fully characterized. In this study, we examined the role of DARPP-32 in gastric cancer cell invasion. Using matrigel-coated Boyden chamber invasion assay, DARPP-32-overexpressing AGS cells showed a three-fold increase in invasion relative to the vector control (P < 0.01). We also tested the transendothelial cell invasion as a measure of cell aggressiveness using the impedance-based human umbilical vein endothelial cells invasion assay and obtained similar results (P < 0.001). Western blot analysis indicated that overexpression of DARPP-32 mediated an increase in the membrane-type 1 matrix metalloproteinase (MT1-MMP) and CXCR4 protein levels. Consistent with the role of MT1-MMP in cleaving extracellular matrix proteins initiating the activation of soluble MMPs, we detected a robust increase in MMP-2 activity in DARPP-32-overexpressing cells. The knockdown of endogenous DARPP-32 in the MKN-45 cells reversed these signaling events and decreased cell invasive activity. We tested whether the invasive activity mediated by DARPP-32 might involve sustained signaling via CXCR4-dependent activation of the MT1-MMP/MMP-2 pathway. The small-molecule CXCR4 antagonist (AMD3100) and CXCR4-siRNA blocked DARPP-32-induced cell invasion. We further examined our hypothesis that DARPP-32 could interact with CXCR4 and stabilize its levels following stimulation with its ligand, CXCL12. Using reciprocal coimmunoprecipitation and immunofluorescence experiments, we found that DARPP-32 and CXCR4 coexist in the same protein complex. DARPP-32 prolonged the CXCR4 protein half-life and reduced ubiquitination of the CXCR4 protein, following treatment with its ligand, CXCL12. In conclusion, these findings show a novel mechanism by which DARPP-32 promotes cell invasion by regulating CXCR4-mediated activation of the MT1-MMP/MMP-2 pathway.

Egr-1 induces DARPP-32 expression in striatal medium spiny neurons via a conserved intragenic element.

DARPP-32 (dopamine and adenosine 3', 5'-cyclic monophosphate cAMP-regulated phosphoprotein, 32 kDa) is a striatal-enriched protein that mediates signaling by dopamine and other first messengers in the medium spiny neurons. The transcriptional mechanisms that regulate striatal DARPP-32 expression remain enigmatic and are a subject of much interest in the efforts to induce a striatal phenotype in stem cells. We report the identification and characterization of a conserved region, also known as H10, in intron IV of the gene that codes for DARPP-32 (Ppp1r1b). This DNA sequence forms multiunit complexes with nuclear proteins from adult and embryonic striata of mice and rats. Purification of proteins from these complexes identified early growth response-1 (Egr-1). The interaction between Egr-1 and H10 was confirmed in vitro and in vivo by super-shift and chromatin immunoprecipitation assays, respectively. Importantly, brain-derived neurotrophic factor (BDNF), a known inducer of DARPP-32 and Egr-1 expression, enhanced Egr-1 binding to H10 in vitro. Moreover, overexpression of Egr-1 in primary striatal neurons induced the expression of DARPP-32, whereas a dominant-negative Egr-1 blocked DARPP-32 induction by BDNF. Together, this study identifies Egr-1 as a transcriptional activator of the Ppp1r1b gene and provides insight into the molecular mechanisms that regulate medium spiny neuron maturation.

Human embryonic stem cell-derived GABA neurons correct locomotion deficits in quinolinic acid-lesioned mice.

Degeneration of medium spiny GABA neurons in the basal ganglia underlies motor dysfunction in Huntington's disease (HD), which presently lacks effective therapy. In this study, we have successfully directed human embryonic stem cells (hESCs) to enriched populations of DARPP32-expressing forebrain GABA neurons. Transplantation of these human forebrain GABA neurons and their progenitors, but not spinal GABA cells, into the striatum of quinolinic acid-lesioned mice results in generation of large populations of DARPP32(+) GABA neurons, which project to the substantia nigra as well as receiving glutamatergic and dopaminergic inputs, corresponding to correction of motor deficits. This finding raises hopes for cell therapy for HD.

Ganglioside GM1 induces phosphorylation of mutant huntingtin and restores normal motor behavior in Huntington disease mice.

Huntington disease (HD) is a progressive neurodegenerative monogenic disorder caused by expansion of a polyglutamine stretch in the huntingtin (Htt) protein. Mutant huntingtin triggers neural dysfunction and death, mainly in the corpus striatum and cerebral cortex, resulting in pathognomonic motor symptoms, as well as cognitive and psychiatric decline. Currently, there is no effective treatment for HD. We report that intraventricular infusion of ganglioside GM1 induces phosphorylation of mutant huntingtin at specific serine amino acid residues that attenuate huntingtin toxicity, and restores normal motor function in already symptomatic HD mice. Thus, our studies have identified a potential therapy for HD that targets a posttranslational modification of mutant huntingtin with critical effects on disease pathogenesis.

Altered DARPP-32 expression in the superior temporal gyrus in schizophrenia.

Many neuroimaging studies have revealed structural abnormalities in the superior temporal gyrus (STG) in schizophrenia (Kasai et al., 2003a, 2003b; Sun et al., 2009). Neurophysiological studies of mismatch negativities (MMN) generated in the STG have suggested impaired function of N-methyl-d-aspartate (NMDA) receptors (Javitt et al., 1996). Although many postmortem studies have been conducted on the pathogenesis of schizophrenia, relatively few reports have studied molecular alterations in the STG (Bowden et al., 2008; Deng and Huang, 2006; Kang et al., 2009; Katsel et al., 2005; Le Corre et al., 2000; Nudmamud and Reynolds, 2001; Sokolov et al., 2000). The STG shows pronounced changes in gene expression when compared to other regions implicated in schizophrenia (Katsel et al., 2005). Dopamine and a cAMP-regulated phosphoprotein of molecular weight 32kDa (DARPP-32) is thought to be closely associated with pathophysiological changes in the dopamine and glutamate systems in schizophrenia because, when activated by phosphorylation, DARPP-32 acts as a critical regulator of D1 dopamine receptor and NMDA receptor activity (Greengard et al., 1999). The molecular pathways involving DARPP-32 appear important in the pathogenesis of schizophrenia. Here, we show dramatic alterations in DARPP-32 expression in the STG of postmortem brains from patients with schizophrenia. To clarify the detailed histological and cellular expression of DARPP-32 in the STG in schizophrenia, we immunohistochemically examined postmortem brains by using specific antibodies. We compared the density of immunoreactive cells of the STG (BA22) from 11 schizophrenia patients with those from 11 age- and sex-matched controls, and found significantly lower densities of DARPP-32-immunoreactive (IR) cells and threonine (Thr) 34-phosphorylated DARPP-32-IR cells in the STG in the schizophrenia group. Thus, the DARPP-32-related pathogenesis in schizophrenia may be more severe in the STG than previously found in the prefrontal cortex.

Altered expression and coregulation of dopamine signalling genes in schizophrenia and bipolar disorder.

INTRODUCTION: signalling through dopamine receptors is of critical importance in the brain and is implicated in schizophrenia and bipolar disorder, but its underlying molecular mechanisms remain poorly understood. MATERIALS AND METHODS: using a yeast two-hybrid approach, we previously identified 11 novel dopamine receptor-interacting proteins. Here we compare gene expression levels for 17 genes [including all 11 dopamine receptor interacting proteins, all 5 dopamine receptors (DRD1-DRD5) and DARPP-32] by real-time polymerase chain reaction, using prefrontal cortex post mortem brain samples from 33 schizophrenic, 32 bipolar disorder and 34 control subjects. RESULTS: the expression of C14ORF28, GNB2L1, MLLT3, DRD2 and DARPP-32 genes was altered in schizophrenia and/or bipolar disorder samples relative to controls (P < 0.05). Hierarchical clustering analysis revealed the expression of these five genes (C14ORF28, GNB2L1, MLLT3, DARPP-32, DRD2) is closely correlated in patients. However, in controls, DRD2 expression in relation to the other genes appears to be very different, suggesting abnormal DRD2 activity is an important trigger in the pathophysiology of schizophrenia and bipolar disorder. CONCLUSIONS: our data suggest: (i) C14ORF28, GNB2L1, MLLT3, DRD2 and DARPP-32 are important in the pathogenesis of schizophrenia and bipolar disorder; (ii) these two disorders share common disease-related mechanisms linked to dopamine signalling; (iii) the expression of these genes is closely correlated; and (iv) DRD2 provides the initial trigger in the pathogenesis of these disorders.

Acute low dose of MK-801 prevents memory deficits without altering hippocampal DARPP-32 expression and BDNF levels in sepsis survivor rats.

Sepsis is characterized by an intense inflammatory reaction with potential neurotoxic effects in the central nervous system and damage to memory and learning ability. We assessed the effects of acute low dose of MK-801 on the memory impairment, hippocampal BDNF levels and DARPP-32 expression ten days after sepsis. Under anesthesia, male Wistar rats underwent either cecal ligation and perforation (CLP) or sham. Then, the animals received either a single systemic injection of MK-801 (0.025 mg/kg) or saline solution. Ten days after CLP, the animals were submitted to the step-down inhibitory avoidance and object recognition tests. Also, the hippocampal BDNF protein levels and DARPP-32 expression were evaluated. MK-801 prevented cognitive impairment, but did not affect the hippocampal BDNF levels. DARPP-32 expression was significantly different only in the animals submitted to sepsis that received MK-801 treatment. Thus, we demonstrated that a single low dose of MK-801 prevented memory impairment without altering hippocampal DARPP-32 expression and BDNF levels.

Phosphorylation of huntingtin at Ser421 in YAC128 neurons is associated with protection of YAC128 neurons from NMDA-mediated excitotoxicity and is modulated by PP1 and PP2A.

YAC transgenic mice expressing poly(Q)-expanded full-length huntingtin (mhtt) recapitulate many behavioral and neuropathological features of Huntington disease (HD). We have previously observed a reduction in phosphorylation of mhtt at S421 in the presence of the mutation for HD. In addition, phosphorylation of normal S421-htt is reduced after excitotoxic stimulation of NMDA receptors (NMDARs). To test whether NMDAR stimulation contributes to reduced pS421-htt levels in HD, we determined phosphorylation of htt at Ser421 after NMDA-induced excitotoxicity in neurons from YAC128 mice. Here, we report that the total level of pS421-htt is reduced in YAC128 primary neurons after excitotoxic NMDAR stimulation. Similarly, the total level of pS421-htt is reduced in YAC128 transgenic mice after quinolinic acid injection into the striatum. In contrast, loss of phosphorylation of pS421-htt is prevented in YAC mice that never develop clinical or neuropathological features of HD [the caspase 6-resistant YAC128 transgene (C6R)]. To gain insight into the mechanisms underlying these findings, we determined that the Ser/Thr protein phosphatases PP1 and PP2A dephosphorylate pS421-htt in situ and after excitotoxic stimulation of NMDARs in neurons. Furthermore, increasing the phosphorylation of htt at S421 by blocking PP1 and PP2A activity protects YAC128 striatal neurons from NMDA-induced cell death. These results, together with the observed modulation of pS421-htt levels by dopamine, the reduced expression of PP1 inhibitor Darpp-32 in the striatum of YAC128 mice, and the reduced phosphorylation of PP1 substrate CreB, point to altered regulation of phosphatase activity in HD and highlight enhancing phosphorylation of htt at S421 as a therapeutic target.

Both t-Darpp and DARPP-32 can cause resistance to trastuzumab in breast cancer cells and are frequently expressed in primary breast cancers.

The clinical use of trastuzumab (Herceptin), a humanized antibody against the HER2 growth factor receptor, has improved survival in patients with breast tumors with ERBB2 amplification and/or over-expression. However, most patients with advanced ERBB2 amplified breast cancers whose tumors initially respond to trastuzumab develop resistance to the drug, leading to tumor progression. To identify factors responsible for acquired resistance to trastuzumab, gene expression profiling was performed on subclones of an ERBB2 amplified breast cancer cell line, BT474, which had acquired resistance to trastuzumab. The most overexpressed gene in these subclones was PPP1R1B, encoding the DARPP-32 phosphatase inhibitor. Western analysis revealed that only the truncated isoform of the DARPP-32 protein, t-Darpp, was overexpressed in the trastuzumab resistant cells. Using gene silencing experiments, we confirmed that t-Darpp over-expression was required for trastuzumab resistance in these cells. Furthermore, transfecting t-Darpp in parental BT-474 cells conferred resistance to trastuzumab, suggesting that t-Darpp expression was sufficient for trastuzumab resistance. We also found that t-Darpp over-expression was associated with Akt activation and that the T75 residue in t-Darpp was required for both Akt activation and trastuzumab resistance. Finally, we found that full-length DARPP-32 and t-Darpp are expressed in a majority of primary breast tumors. Over-expression of full-length DARPP-32 can also confer resistance to trastuzumab and, moreover, is associated with a poor prognostic value in breast cancers. Thus, t-Darpp and DARPP-32 expression are novel prognostic and predictive biomarkers in breast cancer.

The leukocytes expressing DARPP-32 are reduced in patients with schizophrenia and bipolar disorder.

Bipolar disorder (BPD) and schizophrenia (SCZ) are severe disorders representing an enormous social, familiar and individual burden, being SCZ the most disabling psychiatric disorder characterized by psychosis and cognitive impairment. It is well known that SCZ and BPD are associated with abnormalities in dopamine signaling pathway. Recent data in the literature have demonstrated altered expression levels of some proteins involved in the modulation of this pathway in both brain and peripheral tissues. It was shown that protein and mRNA levels of dopamine and cAMP regulated phosphoprotein (DARPP-32) were downregulated in dorsolateral prefrontal cortex (DLPFC) of patients with SCZ or BPD when compared to controls. Due to the difficulty to access brain tissue and the absence of objective laboratory tests for bio-markers, we measured DARPP-32 expression in blood cell sub-populations (CD4+ T lymphocytes, CD56+ NK cells, CD19+ B lymphocytes and CD14+ monocytes) taking advantage of the close relation of nervous and immune systems. Using flow cytometry as the analytical method, our results have shown that the DARPP-32 expression was diminished in CD4+ T lymphocytes, CD19+ B lymphocytes and CD14+ monocytes of BPD patients and was also decreased in CD4+ T lymphocytes and CD56+ NK cells of SCZ patients. These results showed that DARPP-32 expression in immune cells agrees with reports of reduced DARPP-32 protein in the DLPFC of BPD or SCZ patients. Our data suggest that DARPP-32 expression in PBMC could be used as a source of bio-markers to help in the treatment response of neuropsychiatry disorders as a window to the changes in the brain of those patients.

Expression of t-DARPP mediates trastuzumab resistance in breast cancer cells.

PURPOSE: We have investigated the role of t-DARPP in trastuzumab resistance in ERBB2-amplified and overexpressed breast cancer cell lines. EXPERIMENTAL DESIGN: We have used the HR-5 and HR-6 trastuzumab-resistant cells that were established from tumors that recurred in the presence of trastuzumab therapy following xenografts of BT-474 cells in nude mice. In addition, SKBR-3 cells, engineered for stable expression of t-DARPP, and HCC-1569 cells, which have constitutive expression of t-DARPP and are de novo resistant to trastuzumab, were used. RESULTS: We reported > or =15-fold up-regulation of mRNA and protein levels of t-DARPP in HR-5 and HR-6 cells compared with their progenitor BT-474 trastuzumab-sensitive cells. The t-DARPP expression was not regulated by changes in its promoter DNA methylation levels. The SKBR-3 cells stably expressing t-DARPP developed resistance to trastuzumab compared with their parental cells and empty vector controls (P < 0.01). The trastuzumab-resistant cell lines showed a significant increase in pAKT (Ser(473)) and BCL2 protein levels. The small interfering RNA knockdown of t-DARPP in all trastuzumab-resistant cells led to a significant reduction in ERBB2, pAKT (Ser(473)), and BCL2 protein levels with a significant decrease in cell viability (P < or = 0.001) and an increase in cleaved caspase-3 levels, indicating the progression of these cells toward apoptosis. The t-DARPP protein was associated with both heat shock protein 90 and ERBB2 forming a potential protein complex. This association may play a role in regulating ERBB2 protein in trastuzumab-resistant cells. CONCLUSION: We conclude that t-DARPP is a novel molecular target that can mediate the therapeutic resistance to trastuzumab in breast cancer cells.

Reversal of multidrug resistance of adriamycin-resistant gastric adenocarcinoma cells through the up-regulation of DARPP-32.

We have investigated the roles of dopamine and cAMP-regulated phosphoprotein (DARPP-32) in the multidrug resistance (MDR) of gastric cancer cells and the possible underlying mechanisms. The up-regulation of DARPP-32 was found to significantly enhance the sensitivity of cells of human adriamycin (ADR)-resistant gastric adenocarcinoma cell line SGC7901/ADR to vincristine, ADR, 5-fludrouracil and cisplatin. The results of an in vivo drug sensitivity assay confirmed that DARPP-32 may play a specific role in the MDR of gastric cancer. DARPP-32 significantly down-regulated the expression of P-glycoprotein and zinc ribbon domain-containing 1 (ZNRD1), but did not alter the expression of MDR-associated protein or glutathione-S-transferase. The up-regulation of ZNRD1 significantly inhibited the drug sensitivity of gastric cancer cells over-expressing DARPP-32, indicating that ZNRD1 may be important in the DARPP-32-mediated MDR of gastric cancer. DARPP-32 was also able to significantly decrease the anti-apoptotic activity of SGC7901/ADR cells. Further study of the biological functions of DARPP-32 may be helpful for understanding the mechanisms of MDR of gastric cancer cells and developing possible strategies to treat gastric cancer.

DARPP-32 and NCS-1 expression is not altered in brains of rats treated with typical or atypical antipsychotics.

Dopamine-mediated neurotransmission imbalances are associated with several psychiatry illnesses, such as schizophrenia. Recently it was demonstrated that two proteins involved in dopamine signaling are altered in prefrontal cortex (PFC) of schizophrenic patients. DARPP-32 is a key downstream effector of intracellular signaling pathway and is downregulated in PFC of schizophrenic subjects. NCS-1 is a neuronal calcium sensor that can inhibit dopamine receptor D2 internalization and is upregulated in PFC of schizophrenic subjects. It is well known that dopamine D2 receptor is the main target of antipsychotic. Therefore, our purpose was to study if chronic treatment with typical or atypical antipsychotics induced alterations in DARPP-32 and NCS-1 expression in five brain regions: prefrontal cortex, hippocampus, striatum, cortex and cerebellum. We did not find any changes in DARPP-32 and NCS-1 protein expression in any brain region investigated.

DARPP-32 expression in rat brain after electroconvulsive stimulation.

Although electroconvulsive therapy (ECT) has been used as a treatment for mental disorder since 1930s, little progress has been made in the mechanisms underlying its therapeutic or adverse effects. The aim of this work was to analyze the expression of DARPP-32 (a protein with a central role in dopaminergic signaling) in striatum, cortex, hippocampus and cerebellum of Wistar rats subjected to acute or chronic electroconvulsive stimulation (ECS). Rats were submitted to a single stimulation (acute) or to a series of eight stimulations, applied one every 48 h (chronic). Animals were killed for collection of tissue samples at time zero, 0.5, 3, 12, 24 and 48 h after stimulation in the acute model and at the same time intervals after the last stimulation in the chronic model. Our results indicated that acute ECS produces smaller changes in the expression of DARPP-32 but, interestingly, chronic ECS increased transient expression of DARPP-32 in several time frames, in striatum and hippocampus, after the last stimulation. Results on the expression of proteins involved in signaling pathways are relevant for neuropsychiatric disorders and treatment, in particular ECT, and can contribute to shed light on the mechanisms related to therapeutic and adverse effects.

Decreased expression of DARPP-32 in oral premalignant and malignant lesions.

BACKGROUND: DARPP-32 is a neuronal protein that plays a central role in dopaminergic neurotransmission. Although DARPP-32 may contribute to the pathogenesis of several human malignancies, its expression has never been investigated in oral premalignant and malignant lesions. MATERIALS AND METHODS: DARPP-32 expression was examined using immunohistochemistry in 14 normal oral mucosa, 5 normal lower lip mucosa, 41 oral leukoplakia (OL), 30 oral squamous cell carcinoma (OSCC) and 20 lower lip squamous cell carcinoma (LLSCC) specimens. Differences of its expression between groups were analyzed. RESULTS: OSCC and OL with moderate or severe dysplasia showed lower DARPP-32 expression in relation to normal oral mucosa. LLSCC showed lower DARPP-32 expression than normal lower lip mucosa and OSSC. CONCLUSION: The decreased expression of DARPP-32 in oral premalignant and malignant lesions suggests a tumor suppressor role for this protein in the tumorigenesis of these lesions.

AKT and CDK5/p35 mediate brain-derived neurotrophic factor induction of DARPP-32 in medium size spiny neurons in vitro.

Mature striatal medium size spiny neurons express the dopamine and cyclic AMP-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 brain-derived neurotrophic factor (BDNF), retinoic acid, and estrogen. DARPP-32 induction by BDNF in vitro requires phosphatidylinositide 3-kinase (PI3K), but inhibition of phosphorylation of protein kinase B/Akt does not entirely abolish expression of DARPP-32. Moreover, the requirement for Akt has not been established. Using pharmacologic inhibitors of PI3K, Akt, and cyclin-dependent kinase 5 (cdk5) and constitutively active and dominant negative PI3K, Akt, cdk5, and p35 viruses in cultured striatal neurons, we measured BDNF-induced levels of DARPP-32 protein and/or mRNA. We demonstrated that both the PI3K/Akt/mammalian target of rapamycin and the cdk5/p35 signal transduction pathways contribute to the induction of DARPP-32 protein levels by BDNF and that the effects are on both the transcriptional and translational levels. It also appears that PI3K is upstream of cdk5/p35, and its activation can lead to an increase in p35 protein levels. These data support the presence of multiple signal transduction pathways mediating expression of DARPP-32 in vitro, including a novel, important pathway via by which PI3K regulates the contribution of cdk5/p35.

Dopamine receptor signaling molecules are altered in elderly schizophrenic cortex.

Alterations of molecules that mediate dopaminergic signal transduction have been found in schizophrenia, supporting the hypothesis of altered dopaminergic neurotransmission in this illness. To further explore this hypothesis, the authors measured transcript expression of three proteins involved in dopamine (DA) signaling in postmortem dorsolateral prefrontal and anterior cingulate cortex of elderly schizophrenic subjects and a comparison group. The transcript encoding calcyon, a protein that potentiates crosstalk between D1 DA receptors and Gq/11-linked receptors, was increased in schizophrenic prefrontal and cingulate cortex by 25%. Transcript levels of spinophilin, a protein enriched in dendritic spines that modulates excitatory neurotransmission, were increased 22% in dorsolateral prefrontal cortex but were unchanged in anterior cingulate cortex in schizophrenia. Levels of DARPP-32 mRNA, a downstream effector of dopaminergic neurotransmission, were similar in both groups for both cortical groups. These alterations in spinophilin and calcyon mRNA levels in schizophrenic prefrontal and cingulate cortex provide further evidence of altered dopaminergic neurotransmission in this illness.

Development of DARPP-32-positive parts of fetal pig ganglionic eminence and ventral mesencephalon in organotypic slice co-cultures.

Neurons from the fetal pig dopaminergic ventral mesencephalon (VM) and basal ganglia anlage (the ganglionic eminence) were co-cultured as organotypic slice cultures to study the development of the two interconnected brain areas. During a short developmental period (E35-E42), a groove separates the ganglionic eminence into a lateral and a medial part. This was used (a) to study the developmental expression of the striatal marker protein, dopamine and adenosine 3,5-monophosphate regulated phospho-protein (DARPP-32) in the two parts and (b) to compare innervations of the two parts by tyrosine hydroxylase (TH)-positive, dopaminergic fibers from co-cultured slices of the ventral mesencephalon. DARPP-32 expression was more extensive and dense in cultures of the lateral part of the striatal anlage than the medial part. The DARPP-32-positive areas moreover overlapped with areas rich in acetylcholine esterase (AChE) and were the preferred target areas for TH-positive fibers from the co-cultured VM.