Prolonged dopamine D3 receptor stimulation promotes dopamine transporter ubiquitination and degradation through a PKC-dependent mechanism.

The dopamine transporter (DAT) is a membrane glycoprotein in dopaminergic neurons, which modulates extracellular and intracellular dopamine levels. DAT is regulated by different presynaptic proteins, including dopamine D2 (D2R) and D3 (D3R) receptors. While D2R signalling enhances DAT activity, some data suggest that D3R has a biphasic effect. However, despite the extensive therapeutic use of D2R/D3R agonists in neuropsychiatric disorders, this phenomenon has been little studied. In order to shed light on this issue, DAT activity, expression and posttranslational modifications were studied in mice and DAT-D3R-transfected HEK cells. Consistent with previous reports, acute treatment with D2R/D3R agonists promoted DAT recruitment to the plasma membrane and an increase in DA uptake. However, when the treatment was prolonged, DA uptake and total striatal DAT protein declined below basal levels. These effects were inhibited in mice by genetic and pharmacological inactivation of D3R, but not D2R, indicating that they are D3R-dependent. No changes were detected in mesostriatal tyrosine hydroxylase (TH) protein expression and midbrain TH and DAT mRNAs, suggesting that the dopaminergic system is intact and DAT is posttranslationally regulated. The use of immunoprecipitation and cell surface biotinylation revealed that DAT is phosphorylated at serine residues, ubiquitinated and released into late endosomes through a PKCß-dependent mechanism. In sum, the results indicate that long-term D3R activation promotes DAT down-regulation, an effect that may underlie neuroprotective and antidepressant actions described for some D2R/D3R agonists.

Random Lasing Detection of Mutant Huntingtin Expression in Cells.

Huntington's disease (HD) is an autosomal dominant, incurable neurodegenerative disease caused by mutation in the huntingtin gene (HTT). HTT mutation leads to protein misfolding and aggregation, which affect cells' functions and structural features. Because these changes might modify the scattering strength of affected cells, we propose that random lasing (RL) is an appropriate technique for detecting cells that express mutated HTT. To explore this hypothesis, we used a cell model of HD based on the expression of two different forms-pathogenic and non-pathogenic-of HTT. The RL signals from both cell profiles were compared. A multivariate statistical analysis of the RL signals based on the principal component analysis (PCA) and linear discriminant analysis (LDA) techniques revealed substantial differences between cells that expressed the pathogenic and the non-pathogenic forms of HTT.

Inhibition of the mTOR pathway: A new mechanism of ß cell toxicity induced by tacrolimus.

The mechanisms of tacrolimus-induced ß cell toxicity are unknown. Tacrolimus (TAC) and rapamycin (Rapa) both bind to FK506-binding protein 12 (FKBP12). Also, both molecular structures are similar. Because of this similarity, we hypothesized that TAC can also inhibit the mTOR signalling, constituting a possible mechanism of ß cell toxicity. Thus, we studied the effect of TAC and Rapa over the mTOR pathway, v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), and insulin secretion and content in INS-1 ß cells treated with or without glucose and palmitate and in islets from lean or obese rats. TAC and Rapa inhibited the mTOR pathway as reflected by lower levels of phospho-mTOR, phospo-p70S6K, and phospo-S6. The effect of Rapa was larger than TAC. Both drugs reduced the levels of MafA, insulin secretion, and content although these effects were larger with TAC. The changes on MafA and insulin metabolism were observed in cells on glucose and palmitate, in obese animals, and were absent in cells on maintenance medium or in lean animals. In silico docking and immunoprecipitation experiments confirmed that TAC can form a stable noncovalent interaction with FKBP12-mTOR. Thus, the mTOR inhibition may be a mechanism contributing to the diabetogenic effect of TAC.

Long-term controlled GDNF over-expression reduces dopamine transporter activity without affecting tyrosine hydroxylase expression in the rat mesostriatal system.

The dopamine (DA) transporter (DAT) is a plasma membrane glycoprotein expressed in dopaminergic (DA-) cells that takes back DA into presynaptic neurons after its release. DAT dysfunction has been involved in different neuro-psychiatric disorders including Parkinson's disease (PD). On the other hand, numerous studies support that the glial cell line-derived neurotrophic factor (GDNF) has a protective effect on DA-cells. However, studies in rodents show that prolonged GDNF over-expression may cause a tyrosine hydroxylase (TH, the limiting enzyme in DA synthesis) decline. The evidence of TH down-regulation suggests that another player in DA handling, DAT, may also be regulated by prolonged GDNF over-expression, and the possibility that this effect is induced at GDNF expression levels lower than those inducing TH down-regulation. This issue was investigated here using intrastriatal injections of a tetracycline-inducible adeno-associated viral vector expressing human GDNF cDNA (AAV-tetON-GDNF) in rats, and doxycycline (DOX; 0.01, 0.03, 0.5 and 3mg/ml) in the drinking water during 5weeks. We found that 3mg/ml DOX promotes an increase in striatal GDNF expression of 12× basal GDNF levels and both DA uptake decrease and TH down-regulation in its native and Ser40 phosphorylated forms. However, 0.5mg/ml DOX promotes a GDNF expression increase of 3× basal GDNF levels with DA uptake decrease but not TH down-regulation. The use of western-blot under non-reducing conditions, co-immunoprecipitation and in situ proximity ligation assay revealed that the DA uptake decrease is associated with the formation of DAT dimers and an increase in DAT-α-synuclein interactions, without changes in total DAT levels or its compartmental distribution. In conclusion, at appropriate GDNF transduction levels, DA uptake is regulated through DAT protein-protein interactions without interfering with DA synthesis.

Receptor of activated protein C promotes metastasis and correlates with clinical outcome in lung adenocarcinoma.

RATIONALE: Efficient metastasis requires survival and adaptation of tumor cells to stringent conditions imposed by the extracellular milieu. Identification of critical survival signaling pathways in tumor cells might unveil novel targets relevant in disease progression. OBJECTIVES: To investigate the contribution of activated protein C (APC) and its receptor (endothelial protein C receptor [EPCR]) in animal models of lung cancer metastasis and in patients with lung adenocarcinoma. METHODS: Signaling pathway triggered by APC/EPCR and its relevance in apoptosis was studied in vitro. Functional significance was assessed by silencing and blocking antibodies in several in vivo models of lung cancer metastasis in athymic nude Foxn1(nu) mice. We examined EPCR levels using a microarray dataset of 107 patients. Immunohistochemical analysis was performed in an independent cohort of 295 patients with lung adenocarcinoma. MEASUREMENTS AND MAIN RESULTS: The effects of APC binding to EPCR rapidly triggered Akt and extracellular signal-regulated kinase signaling pathways, leading to attenuated in vitro apoptosis. In vivo, silencing of EPCR expression or blocking APC/EPCR interaction reduced infiltration in the target organ, resulting in impaired prometastatic activity. Moreover, overexpression of EPCR induced an increased metastatic activity to target organs. Analysis of clinical samples showed a robust association between high EPCR levels and poor prognosis, particularly in stage I patients. CONCLUSIONS: EPCR and its ligand APC promote cell survival that contributes to tumor cell endurance to stress favoring prometastatic activity of lung adenocarcinoma. EPCR/APC is a novel target of relevance in the clinical outcome of early-stage lung cancer.

DRD3 (dopamine receptor D3) but not DRD2 activates autophagy through MTORC1 inhibition preserving protein synthesis.

Growing evidence shows that autophagy is deficient in neurodegenerative and psychiatric diseases, and that its induction may have beneficial effects in these conditions. However, as autophagy shares signaling pathways with cell death and interferes with protein synthesis, prolonged use of autophagy inducers available nowadays is considered unwise. The search for novel autophagy inducers indicates that DRD2 (dopamine receptor 2)-DRD3 ligands may also activate autophagy, though critical aspects of the action mechanisms and effects of dopamine ligands on autophagy are still unknown. In order to shed light on this issue, DRD2- and DRD3-overexpressing cells and drd2 KO, drd3 KO and wild-type mice were treated with the DRD2-DRD3 agonist pramipexole. The results revealed that pramipexole induces autophagy through MTOR inhibition and a DRD3-dependent but DRD2-independent mechanism. DRD3 activated AMPK followed by inhibitory phosphorylation of RPTOR, MTORC1 and RPS6KB1 inhibition and ULK1 activation. Interestingly, despite RPS6KB1 inhibition, the activity of RPS6 was maintained through activation of the MAPK1/3-RPS6KA pathway, and the activity of MTORC1 kinase target EIF4EBP1 along with protein synthesis and cell viability, were also preserved. This pattern of autophagy through MTORC1 inhibition without suppression of protein synthesis, contrasts with that of direct allosteric and catalytic MTOR inhibitors and opens up new opportunities for G protein-coupled receptor ligands as autophagy inducers in the treatment of neurodegenerative and psychiatric diseases. ABBREVIATIONS: AKT/Protein kinase B: thymoma viral proto-oncogene 1; AMPK: AMP-activated protein kinase; BECN1: beclin 1; EGFP: enhanced green fluorescent protein; EIF4EBP1/4E-BP1: eukaryotic translation initiation factor 4E binding protein 1; GPCR; G protein-coupled receptor; GFP: green fluorescent protein; HEK: human embryonic kidney; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP2K/MEK: mitogen-activated protein kinase kinase; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; MDA: malonildialdehyde; MTOR: mechanistic target of rapamycin kinase; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PPX: pramipexole; RPTOR/raptor: regulatory associated protein of MTOR, complex 1; RPS6: ribosomal protein S6; RPS6KA/p90S6K: ribosomal protein S6 kinase A; RPS6KB1/p70S6K: ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type.

Glutamatergic pallidothalamic projections and their implications in the pathophysiology of Parkinson's disease.

GABAergic projections emitted from the entopeduncular nucleus (ENT) and the substantia nigra pars reticulata (SNr) innervate different thalamic nuclei and they are known to be hyperactive after dopaminergic depletion. Here we show that isoform 2 of the vesicular glutamate transporter (VGLUT2) is expressed by neurons in the ENT nucleus but not in the SNr. Indeed, dual in situ hybridization demonstrated that the ENT nucleus contains two different subpopulations of projection neurons, one single-expressing GAD65/67 mRNAs and another one that co-expresses either of the GAD isoforms together with VGLUT2 mRNA. Unilateral dopaminergic depletion induced marked changes in pallidothalamic-projecting neuron gene expression, resulting in increased expression of GAD65/67 mRNAs together with a clear down-regulation of VGLUT2 mRNA expression. Our results indicate that the increased thalamic inhibition typical of dopamine depletion might be explained by a synergistic effect of increased GABA outflow coupled to decreased glutamate levels, both neurotransmitters coming from ENT neurons.

Pramipexole reduces soluble mutant huntingtin and protects striatal neurons through dopamine D3 receptors in a genetic model of Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by abnormal expansion of the polyglutamine tract in the huntingtin protein (HTT). The toxicity of mutant HTT (mHTT) is associated with intermediate mHTT soluble oligomers that subsequently form intranuclear inclusions. Thus, interventions promoting the clearance of soluble mHTT are regarded as neuroprotective. Striatal neurons are particularly vulnerable in HD. Their degeneration underlies motor symptoms and striatal atrophy, the anatomical hallmark of HD. Recent studies indicate that autophagy may be activated by dopamine D2 and D3 receptor (D2R/D3R) agonists. Since autophagy plays a central role in the degradation of misfolded proteins, and striatal neurons express D2R and D3R, D2R/D3R agonists may promote the clearance of mHTT in striatal neurons. Here, this hypothesis was tested by treating 8-week old R6/1 mice with the D2R/D3R agonist pramipexole for 4weeks. Pramipexole reduced striatal levels of soluble mHTT and increased the size of intranuclear inclusions in R6/1 mice. Furthermore, striatal DARPP-32 levels and motor functions were recovered. These effects were accompanied by an increase in LC3-II and a decrease in p62 in the striatum. Tollip, a selective adaptor of ubiquitinated polyQ proteins to LC3, was also reduced in the striata of R6/1mice but not in their wild-type littermates. No changes were detected in the cerebral cortex where D3R expression is very low, and behavioral and biochemical effects in the striatum were prevented by a D3R antagonist. The findings indicate that PPX protects striatal neurons by promoting the clearance of soluble mHTT through a D3R-mediated mechanism. The evidence of autophagy markers suggests that autophagy is activated, although it is not efficient at removing all mHTT recruited by the autophagic machinery as indicated by the increase in the size of intranuclear inclusions.

miRNA cargo within exosome-like vesicle transfer influences metastatic bone colonization.

Bone metastasis represents one of the most deleterious clinical consequences arising in the context of many solid tumors. Severe osteolysis results from tumor cell colonization of the bone compartment, a process which entails reciprocal exchange of soluble signals between tumor cells and their osseous microenvironment. Recent evidence indicates that tumor-intrinsic miRNAs are pleiotropic regulators of gene expression. But they are also frequently released in exosome-like vesicles (ELV). Yet the functional relevance of the transference of tumor-derived ELV and their miRNA cargo to the extracellular milieu during osseous colonization is unknown. Comparative transcriptomic profiling using an in vivo murine model of bone metastasis identified a repressed miRNA signature associated with high prometastatic activity. Forced expression of single miRNAs identified miR-192 that markedly appeased osseous metastasis in vivo, as shown by X-ray, bioluminescence imaging and microCT scans. Histological examination of metastatic lesions revealed impaired tumor-induced angiogenesis in vivo, an effect that was associated in vitro with decreased hallmarks of angiogenesis. Isolation and characterization of ELV by flow cytometry, Western blot analysis, transmission electron microscopy and nanoparticle tracking analysis revealed the ELV cargo enrichment in miR-192. Consistent with these findings, fluorescent labeled miR-192-enriched-ELV showed the in vitro transfer and release of miR-192 in target endothelial cells and abrogation of the angiogenic program by repression of proangiogenic IL-8, ICAM and CXCL1. Moreover, in vivo infusion of fluorescent labeled ELV efficiently targeted cells of the osseous compartment. Furthermore, treatment with miR-192 enriched ELV in a model of in vivo bone metastasis pre-conditioned osseous milieu and impaired tumor-induced angiogenesis, thereby reducing the metastatic burden and tumor colonization. Changes in the miRNA-cargo content within ELV represent a novel mechanism heavily influencing bone metastatic colonization, which is most likely relevant in other target organs. Mechanistic mimicry of this phenomenon by synthetic nanoparticles could eventually emerge as a novel therapeutic approach.

RHOB influences lung adenocarcinoma metastasis and resistance in a host-sensitive manner.

Lung adenocarcinoma (ADC) is the most common lung cancer subtype and presents a high mortality rate. Clinical recurrence is often associated with the emergence of metastasis and treatment resistance. The purpose of this study was to identify genes with high prometastatic activity which could potentially account for treatment resistance. Global transcriptomic profiling was performed by robust microarray analysis in highly metastatic subpopulations. Extensive in vitro and in vivo functional studies were achieved by overexpression and by silencing gene expression. We identified the small GTPase RHOB as a gene that promotes early and late stages of metastasis in ADC. Gene silencing of RHOB prevented metastatic activity in a systemic murine model of bone metastasis. These effects were highly dependent on tumor-host interactions. Clinical analysis revealed a marked association between high RHOB levels and poor survival. Consistently, high RHOB levels promote metastasis progression, taxane-chemoresistance, and contribute to the survival advantage to γ-irradiation. We postulate that RHOB belongs to a novel class of "genes of recurrence" that have a dual role in metastasis and treatment resistance.

Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) reduces cell survival, homing, and colonization in lung cancer bone metastasis.

PURPOSE: We investigated the role of the collagen-binding receptor discoidin domain receptor-1 (DDR1) in the initiation and development of bone metastasis. EXPERIMENTAL DESIGN: We conducted immunohistochemical analyses in a cohort of 83 lung cancer specimens and examined phosphorylation status in a panel of human lung cancer cell lines. Adhesion, chemotaxis, invasiveness, metalloproteolytic, osteoclastogenic, and apoptotic assays were conducted in DDR1-silenced cells. In vivo, metastatic osseous homing and colonization were assessed in a murine model of metastasis. RESULTS: DDR1 was expressed in a panel of human lung cancer cell lines, and high DDR1 levels in human lung tumors were associated with poor survival. Knockdown (shDDR1) cells displayed unaltered growth kinetics in vitro and in vivo. In contrast, shDDR1 cells showed reduced invasiveness in collagen matrices and increased apoptosis in basal conditions and induced apoptosis in vitro. More importantly, conditioned media of DDR1-knockdown cells decreased osteoclastogenic activity in vitro. Consequently, in a model of tumor metastasis to bone, lack of DDR1 showed decreased metastatic activity associated with reduced tumor burden and osteolytic lesions. These effects were consistent with a substantial reduction in the number of cells reaching the bone compartment. Moreover, intratibial injection of shDDR1 cells significantly decreased bone tumor burden, suggesting impaired colonization ability that was highly dependent on the bone microenvironment. CONCLUSIONS: Disruption of DDR1 hampers tumor cell survival, leading to impaired early tumor-bone engagement during skeletal homing. Furthermore, inhibition of DDR1 crucially alters bone colonization. We suggest that DDR1 represents a novel therapeutic target involved in bone metastasis.

Tumor-stromal interactions of the bone microenvironment: in vitro findings and potential in vivo relevance in metastatic lung cancer models.

Lung cancer comprises a large variety of histological subtypes with a frequent proclivity to form bone metastasis; a condition associated with dismal prognosis. To identify common mechanisms in the development of osteolytic metastasis, we systematically screened a battery of lung cancer cell lines and developed three models of non-small cell lung cancer (NSCLC) with a common proclivity to form osseous lesions, which represented different histological subtypes. Comparative analysis revealed different incidences and latency times. These differences were correlated with cell-type-specific secretion of osteoclastogenic factors, including macrophage inflammatory protein-1α, interleukin-8 and parathyroid hormone-related protein, some of which were exacerbated in conditions that mimicked tumor-stroma interactions. In addition, a distinct signature of matrix metalloproteinase (MMP) activity derived from reciprocal tumor-stroma interactions was detected for each tumor cell line. Thus, these results suggest subtle differences in the mechanisms of bone colonization for each lung cancer subtype, but share, although each to a different degree, dual MMP and osteoclastogenic activities that are differentially enhanced upon tumor-stromal interactions.

PDGFR signaling blockade in marrow stroma impairs lung cancer bone metastasis.

Bone microenvironment and cell-cell interactions are crucial for the initiation and development of metastasis. By means of a pharmacologic approach, using the multitargeted tyrosine kinase inhibitor sunitinib, we tested the relevance of the platelet-derived growth factor receptor (PDGFR) axis in the bone marrow (BM) stromal compartment for the initiation and development of lung cancer metastasis to bone. PDGFRß was found to be the main tyrosine kinase target of sunitinib expressed in BM stromal ST-2 and MC3T3-E1 preosteoblastic cells. In contrast, no expression of sunitinib-targeted receptors was found in A549M1 and low levels in H460M5 lung cancer metastatic cells. Incubation of ST-2 and human BM endothelial cells with sunitinib led to potent cell growth inhibition and induction of apoptosis in a dose-dependent manner. Similarly, sunitinib induced a robust proapoptotic effect in vivo on BM stromal PDGFRß(+) cells and produced extensive disruption of tissue architecture and vessel leakage in the BM cavity. Pretreatment of ST-2 cells with sunitinib also hindered heterotypic adhesion to lung cancer cell lines. These effects were correlated with changes in cell-cell and cell-matrix molecules in both stromal and tumor cells. Pretreatment of mice with sunitinib before intracardiac inoculation of A549M1 or H460M5 cells caused marked inhibition of tumor cells homing to bone, whereas no effect was found when tumor cells were pretreated before inoculation. Treatment with sunitinib dramatically increased overall survival and prevented tumor colonization but not bone lesions, whereas combination with zoledronic acid resulted in marked reduction of osteolytic lesions and osseous tumor burden. Thus, disruption of the PDGFR axis in the BM stroma alters heterotypic tumor-stromal and tumor-matrix interactions, thereby preventing efficient engagement required for bone homing and osseous colonization. These results support the notion that concomitant targeting of the tumor and stromal compartment is a more effective approach for blocking bone metastasis.

A novel lung cancer signature mediates metastatic bone colonization by a dual mechanism.

Bone is a frequent target of lung cancer metastasis, which is associated with significant morbidity and a dismal prognosis. To identify and functionally characterize genes involved in the mechanisms of osseous metastasis, we developed a murine lung cancer model. Comparative transcriptomic analysis identified genes encoding signaling molecules (such as TCF4 and PRKD3) and cell anchorage-related proteins (MCAM and SUSD5), some of which were basally modulated by transforming growth factor-beta (TGF-beta) in tumor cells and in conditions mimicking tumor-stromal interactions. Triple gene combinations induced not only high osteoclastogenic activity but also a marked enhancement of global metalloproteolytic activities in vitro. These effects were strongly associated with robust bone colonization in vivo, whereas this gene subset was ineffective in promoting local tumor growth and cell homing activity to bone. Interestingly, global inhibition of metalloproteolytic activities and simultaneous TGF-beta blockade in vivo led to increased survival and a remarkable attenuation of bone tumor burden and osteolytic metastasis. Thus, this metastatic gene signature mediates bone matrix degradation by a dual mechanism of induction of TGF-beta-dependent osteoclastogenic bone resorption and enhancement of stroma-dependent metalloproteolytic activities. Our findings suggest the cooperative contribution of host-derived and cell autonomous effects directed by a small subset of genes in mediating aggressive osseous colonization.