RESUMO
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.
Assuntos
Agonistas de Dopamina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Proteína Quinase C/metabolismo , Proteólise/efeitos dos fármacos , Receptores de Dopamina D3/metabolismo , Ubiquitinação/fisiologia , Animais , Relação Dose-Resposta a Droga , Esquema de Medicação , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pramipexol/farmacologia , Receptores de Dopamina D3/agonistas , Ubiquitinação/efeitos dos fármacosRESUMO
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.
Assuntos
Doença de Huntington , Doenças Neurodegenerativas , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , MutaçãoRESUMO
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.