RESUMO
Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG nucleotide expansion, which encodes the amino acid glutamine, in the huntingtin gene. HD is characterized by motor, cognitive, and psychiatric dysfunctions. In a previous study, we showed by qPCR that some genes altered in an HD mouse model were also altered in blood of HD patients. These alterations were mainly with respect to the dynein family. Therefore, this study aimed to investigate whether dynein light chain Tctex type 1 (DYNLT1) is altered in HD patients and if there is a correlation between DYNLT1 gene expression changes and disease progression. We assessed the DYNLT1 gene expression in the blood of 19 HD patients and 20 healthy age-matched controls. Also, in 6 of these patients, we analyzed the DYNLT1 expression at two time points, 3 years apart. The DYNLT1 gene expression in the whole blood of HD patients was significantly downregulated and this difference was widened in later stages. These data suggest that DYNLT1 could emerge as a peripheral prognostic indicator in HD and, also, might be a target for potential intervention in the future.
Assuntos
Dineínas/genética , Doença de Huntington , Animais , Estudos de Casos e Controles , Modelos Animais de Doenças , Progressão da Doença , Dineínas/sangue , Expressão Gênica , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , CamundongosRESUMO
Synthesis of acetylcholine depends on the plasma membrane uptake of choline by a high affinity choline transporter (CHT1). Choline uptake is regulated by nerve impulses and trafficking of an intracellular pool of CHT1 to the plasma membrane may be important for this regulation. We have generated a hemagglutinin (HA) epitope tagged CHT1 to investigate the organelles involved with intracellular trafficking of this protein. Expression of CHT1-HA in HEK 293 cells establishes Na+-dependent, hemicholinium-3 sensitive high-affinity choline transport activity. Confocal microscopy reveals that CHT1-HA is found predominantly in intracellular organelles in three different cell lines. Importantly, CHT1-HA seems to be continuously cycling between the plasma membrane and endocytic organelles via a constitutive clathrin-mediated endocytic pathway. In a neuronal cell line, CHT1-HA colocalizes with the early endocytic marker green fluorescent protein (GFP)-Rab 5 and with two markers of synaptic-like vesicles, VAMP-myc and GFP-VAChT, suggesting that in cultured cells CHT1 is present mainly in organelles of endocytic origin. Subcellular fractionation and immunoisolation of organelles from rat brain indicate that CHT1 is present in synaptic vesicles. We propose that intracellular CHT1 can be recruited during stimulation to increase choline uptake in nerve terminals.
Assuntos
Clatrina/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Hemicolínio 3/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Humanos , Rim/citologia , Rim/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Proteínas R-SNARE , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sinaptossomos/metabolismo , Proteínas Vesiculares de Transporte de AcetilcolinaRESUMO
Synaptic vesicle proteins are suggested to travel from the trans-Golgi network to active zones via tubulovesicular organelles, but the participation of different populations of endosomes in trafficking remains a matter of debate. Therefore, we generated a green fluorescent protein (GFP)-tagged version of the vesicular acetylcholine transporter (VAChT) and studied the localization of VAChT in organelles in the cell body and varicosities of living cholinergic cells. GFP-VAChT is distributed to both early and recycling endosomes in the cell body and is also observed to accumulate in endocytic organelles within varicosities of SN56 cells. GFP-VAChT positive organelles in varicosities are localized close to plasma membrane and are labeled with FM4-64 and GFP-Rab5, markers of endocytic vesicles and early endosomes, respectively. A GFP-VAChT mutant lacking a dileucine endocytosis motif (leucine residues 485 and 486 changed to alanine residues) accumulated at the plasma membrane in SN56 cells. This endocytosis-defective GFP-VAChT mutant is localized primarily at the somal plasma membrane and exhibits reduced neuritic targeting. Furthermore, the VAChT mutant did not accumulate in varicosities, as did VAChT. Our data suggest that clathrin-mediated internalization of VAChT to endosomes at the cell body might be involved in proper sorting and trafficking of VAChT to varicosities. We conclude that genesis of competent cholinergic secretory vesicles depends on multiple interactions of VAChT with endocytic proteins.
Assuntos
Acetilcolina/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/farmacocinética , Proteínas de Membrana Transportadoras , Neurônios/metabolismo , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Animais , Vesículas Revestidas por Clatrina/metabolismo , Endocitose/fisiologia , Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde , Indicadores e Reagentes/farmacocinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/farmacocinética , Dados de Sequência Molecular , Mutagênese/fisiologia , Neurônios/citologia , Transmissão Sináptica/fisiologia , Transfecção , Células Tumorais Cultivadas , Proteínas Vesiculares de Transporte de AcetilcolinaRESUMO
G protein-coupled receptor (GPCR) activation is followed rapidly by adaptive changes that serve to diminish the responsiveness of a cell to further stimulation. This process, termed desensitization, is the consequence of receptor phosphorylation, arrestin binding, sequestration and down-regulation. GPCR phosphorylation is initiated within seconds to minutes of receptor activation and is mediated by both second messenger-dependent protein kinases and receptor-specific G protein-coupled receptor kinases (GRKs). Desensitization in response to GRK-mediated phosphorylation involves the binding of arrestin proteins that serve to sterically uncouple the receptor from its G protein. GPCR sequestration, the endocytosis of receptors to endosomes, not only contributes to the temporal desensitization of GPCRs, but plays a critical role in GPCR resensitization. GPCR down-regulation, a loss of the total cellular complement of receptors, is the consequence of both increased lysosomal degradation and decreased mRNA synthesis of GPCRs. While each of these agonist-mediated desensitization processes are initiated within a temporally dissociable time frame, recent data suggest that they are intimately related to one another. The use of green fluorescent protein from the jellyfish Aqueora victoria as an epitope tag with intrinsic fluorescence has facilitated our understanding of the relative relationship between GRK phosphorylation, arrestin binding, receptor sequestration and down-regulation.