RESUMO
Small molecules that increase the presynaptic function of aminergic cells may provide neuroprotection in Parkinson's disease (PD) as well as treatments for attention deficit hyperactivity disorder (ADHD) and depression. Model genetic organisms such as Drosophila melanogaster may enhance the detection of new drugs via modifier or 'enhancer/suppressor' screens, but this technique has not been applied to processes relevant to psychiatry. To identify new aminergic drugs in vivo, we used a mutation in the Drosophila vesicular monoamine transporter (dVMAT) as a sensitized genetic background and performed a suppressor screen. We fed dVMAT mutant larvae â¼ 1000 known drugs and quantitated rescue (suppression) of an amine-dependent locomotor deficit in the larva. To determine which drugs might specifically potentiate neurotransmitter release, we performed an additional secondary screen for drugs that require presynaptic amine storage to rescue larval locomotion. Using additional larval locomotion and adult fertility assays, we validated that at least one compound previously used clinically as an antineoplastic agent potentiates the presynaptic function of aminergic circuits. We suggest that structurally similar agents might be used to development treatments for PD, depression and ADHD, and that modifier screens in Drosophila provide a new strategy to screen for neuropsychiatric drugs. More generally, our findings demonstrate the power of physiologically based screens for identifying bioactive agents for select neurotransmitter systems.
Assuntos
Antiparkinsonianos/farmacologia , Drosophila melanogaster , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Animais , Animais Geneticamente Modificados , Dacarbazina/farmacologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Fertilidade/efeitos dos fármacos , Larva/efeitos dos fármacos , Larva/fisiologia , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Mutação , Doença de Parkinson/tratamento farmacológico , Pergolida/farmacologia , Sinapses/efeitos dos fármacos , Proteínas Vesiculares de Transporte de Monoamina/genéticaRESUMO
Many psychoactive drugs influence the transport of neurotransmitters across biological membranes, suggesting that the physiological regulation of neurotransmitter transport might contribute to normal and perhaps abnormal behaviour. Over the past few years, molecular characterization of the neurotransmitter transporters has enabled investigation of their subcellular location and regulation. The analysis of location suggests that membrane trafficking has an important role in the normal function of these proteins. One of the major regulatory mechanisms also involves changes in localization that might contribute to synaptic plasticity. This article discusses recent work on the membrane trafficking of neurotransmitter transporters and its role in regulating their activity.
Assuntos
Proteínas de Transporte/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurotransmissores/metabolismo , Transmissão Sináptica/fisiologia , Afeto/fisiologia , Animais , Transporte Biológico , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/classificação , Células Cultivadas , Cães , Humanos , Drogas Ilícitas/farmacologia , Transporte de Íons , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos , Camundongos Mutantes Neurológicos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Neurônios/metabolismo , Neurônios/ultraestrutura , Células PC12 , Ratos , Transdução de Sinais , SuínosRESUMO
The release of biogenic amines from large dense core vesicles (LDCVs) depends on localization of the vesicular monoamine transporter VMAT2 to LDCVs. We now find that a cluster of acidic residues including two serines phosphorylated by casein kinase 2 is required for the localization of VMAT2 to LDCVs. Deletion of the acidic cluster promotes the removal of VMAT2 from LDCVs during their maturation. The motif thus acts as a signal for retention on LDCVs. In addition, replacement of the serines by glutamate to mimic phosphorylation promotes the removal of VMAT2 from LDCVs, whereas replacement by alanine to prevent phosphorylation decreases removal. Phosphorylation of the acidic cluster thus appears to reduce the localization of VMAT2 to LDCVs by inactivating a retention mechanism.
Assuntos
Motivos de Aminoácidos , Monoaminas Biogênicas/metabolismo , Exocitose/fisiologia , Glicoproteínas , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana , Proteínas de Membrana Transportadoras , Neuropeptídeos , Sinais Direcionadores de Proteínas , Transporte Proteico/fisiologia , Vesículas Secretórias/metabolismo , Sequência de Aminoácidos , Animais , Monoaminas Biogênicas/química , Brefeldina A/farmacologia , Proteínas de Transporte/metabolismo , Fracionamento Celular , Cromograninas , Immunoblotting , Glicoproteínas de Membrana/genética , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Células PC12 , Fosforilação , Inibidores da Síntese de Proteínas/farmacologia , Proteínas/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vesículas Secretórias/química , Vesículas Secretórias/efeitos dos fármacos , Proteínas Vesiculares de Transporte de Aminas Biogênicas , Proteínas Vesiculares de Transporte de Monoamina , Proteínas de Transporte Vesicular , Rede trans-Golgi/metabolismoRESUMO
Vesicular transport proteins package classical neurotransmitters for regulated exocytotic release, and localize to at least two distinct types of secretory vesicles. In PC12 cells, the vesicular acetylcholine transporter (VAChT) localizes preferentially to synaptic-like microvesicles (SLMVs), whereas the closely related vesicular monoamine transporters (VMATs) localize preferentially to large dense core vesicles (LDCVs). VAChT and the VMATs contain COOH-terminal, cytoplasmic dileucine motifs required for internalization from the plasma membrane. We now show that VAChT undergoes regulated phosphorylation by protein kinase C on a serine (Ser-480) five residues upstream of the dileucine motif. Replacement of Ser-480 by glutamate, to mimic the phosphorylation event, increases the localization of VAChT to LDCVs. Conversely, the VMATs contain two glutamates upstream of their dileucine-like motif, and replacement of these residues by alanine conversely reduces sorting to LDCVs. The results provide some of the first information about sequences involved in sorting to LDCVs. Since the location of the transporters determines which vesicles store classical neurotransmitters, a change in VAChT trafficking due to phosphorylation may also influence the mode of transmitter release.
Assuntos
Proteínas de Transporte/metabolismo , Grânulos Citoplasmáticos/fisiologia , Grânulos Citoplasmáticos/ultraestrutura , Proteínas de Membrana Transportadoras , Neuropeptídeos , Proteínas de Transporte Vesicular , Substituição de Aminoácidos , Animais , Células COS , Proteínas de Transporte/química , Proteínas de Transporte/genética , Fracionamento Celular , Membrana Celular/metabolismo , Centrifugação com Gradiente de Concentração , Clonagem Molecular , Ácido Glutâmico , Leucina , Glicoproteínas de Membrana/metabolismo , Mutagênese Sítio-Dirigida , Células PC12 , Fosforilação , Mutação Puntual , Proteína Quinase C/metabolismo , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina , Serotonina/metabolismo , Vesículas Sinápticas/fisiologia , Vesículas Sinápticas/ultraestrutura , Proteínas Vesiculares de Transporte de Acetilcolina , Proteínas Vesiculares de Transporte de Aminas BiogênicasRESUMO
While the transporters that accumulate classical neurotransmitters in synaptic vesicles have been identified, little is known about how their expression regulates synaptic transmission. We have used adenoviral-mediated transfection to increase expression of the brain vesicular monoamine transporter VMAT2 and presynaptic amperometric recordings to characterize the effects on quantal release. In presynaptic axonal varicosities of ventral midbrain neurons in postnatal culture, VMAT2 overexpression in small synaptic vesicles increased both quantal size and frequency, consistent with the recruitment of synaptic vesicles that do not normally release dopamine. This was confirmed using noncatecholaminergic AtT-20 cells, in which VMAT2 expression induced the quantal release of dopamine. The ability to increase quantal size in vesicles that were already competent for dopamine release was shown in PC12 cells, in which VMAT2 expression increased the quantal size but not the number of release events. These results demonstrate that vesicle transporters limit the rate of transmitter accumulation and can alter synaptic strength through two distinct mechanisms.
Assuntos
Glicoproteínas de Membrana/biossíntese , Proteínas de Membrana Transportadoras , Neuropeptídeos , Vesículas Sinápticas/metabolismo , Adenoviridae/genética , Animais , Axônios/metabolismo , Células Cultivadas , Dopamina/metabolismo , Eletroquímica , Exocitose , Glicoproteínas de Membrana/genética , Camundongos , Microeletrodos , Modelos Neurológicos , Neurônios/citologia , Neurônios/metabolismo , Fenótipo , Distribuição de Poisson , Terminações Pré-Sinápticas/metabolismo , RNA Mensageiro/biossíntese , Ratos , Transmissão Sináptica/fisiologia , Transfecção , Tirosina 3-Mono-Oxigenase/genética , Proteínas Vesiculares de Transporte de Aminas Biogênicas , Proteínas Vesiculares de Transporte de MonoaminaRESUMO
Studies assessing the effect of glucocorticoids on beta-endorphin regulation in man have yielded inconsistent results. As measured by a highly specific beta-endorphin assay procedure, plasma immunoreactive beta-endorphin (irB-EP) in six healthy subjects was significantly suppressed, along with cortisol, after a 2 mg oral dose of dexamethasone (DEX) (pre-DEX mean +/- S.E.M. in pg/ml = 15.3 +/- 2.0, post-DEX = 9.1 +/- 0,5 t = 3.46, p less than 0.01) but not after placebo (pre-placebo = 17.8 +/- 2.9, post-placebo = 17.2 +/- 1.5, t = 0.27). A less specific B-EP assay system, which yielded spurious irB-EP values (102 +/- 2.3 pg/ml) in plasma stripped of B-EP silicic acid, did not detect this DEX-induced change in plasma irB-EP levels (pre-DEX = 119 +/- 9.3, post-DEX = 112 +/- 5.0, t = 0.64; pre-placebo = 118 +/- 6.4, post-placebo = 119 +/- 4.3, t = 0.32). The methodological artifact encountered in this relatively non-specific assay system appears to account for the failure of an earlier study to demonstrate DEX suppression of irB-EP in man.
Assuntos
Dexametasona/farmacologia , Endorfinas/sangue , Adulto , Idoso , Método Duplo-Cego , Humanos , Hidrocortisona/sangue , Masculino , Pessoa de Meia-Idade , Radioimunoensaio , beta-EndorfinaRESUMO
We have established a new simple behavioral paradigm in Drosophila melanogaster to determine how genes and the environment influence the behavior of flies within a social group. Specifically, we measure social space as the distance between two flies. The majority of Canton-s flies, regardless of their gender, are within two body lengths from each other. Their social experience affects this behavior, with social isolation reducing and mating enhancing social space respectively, in both males and females. Unlike several other social behaviors in the fly, including the formation of social groups themselves (a well-described behavior), social space does not require the perception of the previously identified aggregation pheromone cis-vaccenyl acetate. Conversely, performance of the assay in darkness or mutations in the eye pigmentation gene white increased social space. Our results establish a new assay for the genetic dissection of a fundamental mode of social interaction.
Assuntos
Drosophila , Comportamento Social , Animais , Feminino , Masculino , Meio SocialRESUMO
The synaptic machinery for neurotransmitter storage is cell-type specific. Although most elements of biosynthesis and transport have been identified, it remains unclear whether additional factors may be required to maintain this specificity. The Drosophila serotonin transporter (dSERT) is normally expressed exclusively in serotonin (5-HT) neurons in the CNS. Here we examine the effects of ectopic transcriptional expression of dSERT in the Drosophila larval CNS. We find a surprising limitation on 5-HT storage following ectopic expression of dSERT and green fluorescence protein-tagged dSERT (GFP-dSERT). When dSERT transcription is driven ectopically in the CNS, 5-HT is detectable only in 5-HT, dopamine (DA), and a very limited number of additional neurons. Addition of exogenous 5-HT does not dramatically broaden neuronal storage sites, so this limitation is only partly due to restricted intercellular diffusion of 5-HT. Furthermore, this limitation is not due to gross mislocalization of dSERT, because cells lacking or containing 5-HT show similar levels and subcellular distribution of GFP-dSERT protein, nor is it due to lack of the vesicular transporter, dVMAT. These data suggest that a small number of neurons selectively express factor(s) required for 5-HT storage, and potentially for function of dSERT.
Assuntos
Sistema Nervoso Central/crescimento & desenvolvimento , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Neurônios/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Serotonina/metabolismo , Animais , Compartimento Celular/efeitos dos fármacos , Compartimento Celular/fisiologia , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Dopamina/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Fluorescência Verde , Larva/citologia , Larva/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/fisiologia , Serotonina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Vesículas Transportadoras/metabolismoRESUMO
Aminergic signaling pathways have been implicated in a variety of neuropsychiatric illnesses, but the mechanisms by which these pathways influence complex behavior remain obscure. Vesicular monoamine transporters (VMATs) have been shown to regulate the amount of monoamine neurotransmitter that is stored and released from synaptic vesicles in mammalian systems, and an increase in their expression has been observed in bipolar patients. The model organism Drosophila melanogaster provides a powerful, but underutilized genetic system for studying how dopamine (DA) and serotonin (5HT) may influence behavior. We show that a Drosophila isoform of VMAT (DVMAT-A) is expressed in both dopaminergic and serotonergic neurons in the adult Drosophila brain. Overexpression of DVMAT-A in these cells potentiates stereotypic grooming behaviors and locomotion and can be reversed by reserpine, which blocks DVMAT activity, and haloperidol, a DA receptor antagonist. We also observe a prolongation of courtship behavior, a decrease in successful mating and a decrease in fertility, suggesting a role for aminergic circuits in the modulation of sexual behaviors. Finally, we find that DMVAT-A overexpression decreases the fly's sensitivity to cocaine, suggesting that the synaptic machinery responsible for this behavior may be downregulated. DVMAT transgenes may be targeted to additional neuronal pathways using standard Drosophila techniques, and our results provide a novel paradigm to study the mechanisms by which monoamines regulate complex behaviors relevant to neuropsychiatric illness.
Assuntos
Cocaína/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Atividade Motora/fisiologia , Proteínas Vesiculares de Transporte de Monoamina/genética , Animais , Animais Geneticamente Modificados , Comportamento Animal/efeitos dos fármacos , Corte , Dopamina/fisiologia , Proteínas de Drosophila/metabolismo , Feminino , Expressão Gênica , Masculino , Serotonina/fisiologia , Proteínas Vesiculares de Transporte de Monoamina/metabolismoRESUMO
Drosophila chaoptin, required for photoreceptor cell morphogenesis, is a member of the leucine-rich repeat family of proteins. On the basis of biochemical and genetic analyses we previously proposed that chaoptin might function as a cell adhesion molecule. To test this hypothesis, chaoptin cDNA driven by the hsp 70 promoter was transfected into non-self-adherent Drosophila Schneider line 2 (S2) cells. Following heat shock induction of chaoptin expression, the transfected S2 cells formed multicellular aggregates. Mixing experiments of chaoptin expressing and non-expressing cells suggest that chaoptin expressing cells adhere homotypically. Previously it was shown that chaoptin is exclusively localized to photoreceptor cells. Thus, chaoptin is a cell-type-specific adhesion molecule. Biochemical analyses presented in this paper demonstrate that chaoptin is linked to the extracellular surface of the plasma membrane by covalent attachment to glycosyl-phosphatidylinositol. We propose that chaoptin and several other members of the leucine-rich repeat family of proteins define a new class of cell adhesion molecules.
Assuntos
Drosophila/genética , Glicoproteínas de Membrana/genética , Família Multigênica , Células Fotorreceptoras/metabolismo , Animais , Adesão Celular , Agregação Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , DNA/genética , Drosophila/ultraestrutura , Proteínas de Drosophila , Glicolipídeos/fisiologia , Leucina/genética , Glicoproteínas de Membrana/imunologia , Sensibilidade e Especificidade , Fatores de Tempo , Fosfolipases Tipo C/farmacologiaRESUMO
Monoclonal antibody 24B10 (MAb24B10) specifically stains photoreceptor neurons in D. melanogaster. It recognizes a 160 kd glycoprotein localized to the extracellular face of the plasma membrane. Using an immunoscreen, we identified two mutations in the encoding gene that cause microvillar disorganization in developing rhabdomeres and disruption of the closely apposed membranes of adjacent cells. In accordance with the mutant phenotype, we have renamed this genetic locus chaoptic and the encoded glycoprotein, chaoptin. Immunoelectron microscopy indicates that chaoptin is distributed along the length of the microvillus. This localization and the morphological abnormalities in mutants support the hypothesis that chaoptin may mediate adhesion between closely apposed membranes. In principle, the immunoscreen utilized here can be used to identify mutations in any gene in Drosophila for which antibodies to the gene product are available.
Assuntos
Drosophila melanogaster/genética , Glicoproteínas de Membrana/fisiologia , Células Fotorreceptoras/ultraestrutura , Animais , Diferenciação Celular , Membrana Celular/ultraestrutura , DNA/análise , Proteínas de Drosophila , Drosophila melanogaster/anatomia & histologia , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Glicoproteínas/análise , Glicoproteínas/genética , Glicoproteínas/fisiologia , Técnicas Imunoenzimáticas , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/genética , Microscopia Eletrônica , Microvilosidades/ultraestrutura , Mutação , Hibridização de Ácido Nucleico , Células Fotorreceptoras/análise , Células Fotorreceptoras/fisiologia , RNA/análise , RNA Mensageiro/análiseRESUMO
Chaoptin is a photoreceptor cell-specific membrane protein. Analysis of chaoptin mutants demonstrates that this glycoprotein plays a critical role in photoreceptor cell morphogenesis. We have deduced chaoptin's primary structure from the cDNA sequence and examined its membrane topology. Chaoptin is largely composed of 41 potentially amphipathic repeats. Remarkably homologous repeats have been reported in both yeast and human, suggesting their general importance as a structural and/or functional motif. Chaoptin synthesized in vitro is associated with rough endoplasmic reticulum microsomal membrane in an integral fashion, consistent with its extraction characteristics in vivo. The resistance to proteolytic digestion of in vitro synthesized and processed chaoptin suggests that it is primarily localized to the extracellular leaflet of the lipid bilayer. These data are consistent with the proposal that chaoptin is involved in the adhesion between photoreceptor cell membranes.
Assuntos
Drosophila melanogaster/genética , Glicoproteínas de Membrana/genética , Células Fotorreceptoras/fisiologia , Sequências Repetitivas de Ácido Nucleico , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/genética , DNA Recombinante , Proteínas de Drosophila , Drosophila melanogaster/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Leucina , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microssomos/metabolismo , Dados de Sequência Molecular , Morfogênese , Células Fotorreceptoras/ultraestrutura , Biossíntese de Proteínas , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Homologia de Sequência do Ácido NucleicoRESUMO
Specific transport proteins mediate the packaging of neurotransmitters into secretory vesicles and consequently require targeting to the appropriate intracellular compartment. To identify residues in the neuron-specific vesicular monoamine transporter (VMAT2) responsible for endocytosis, we examined the effect of amino (NH2-) and carboxyl (COOH-)-terminal mutations on steady state distribution and internalization. Deletion of a critical COOH-terminal domain sequence (AKEEKMAIL) results in accumulation of VMAT2 at the plasma membrane and a 50% reduction in endocytosis. Site-directed mutagenesis shows that replacement of the isoleucine-leucine pair within this sequence by alanine-alanine alone reduces endocytosis by 50% relative to wild type VMAT2. Furthermore, the KEEKMAIL sequence functions as an internalization signal when transferred to the plasma membrane protein Tac, and the mutation of the isoleucine-leucine pair also abolishes internalization of this protein. The closely related vesicular acetylcholine transporter (VAChT) contains a similar di-leucine sequence within the cytoplasmic COOH-terminal domain that when mutated results in accumulation of VAChT at the plasma membrane. The VAChT di-leucine sequence also confers internalization when appended to two other proteins and in one of these chimeras, conversion of the di-leucine sequence to di-alanine reduces the internalization rate by 50%. Both VMAT2 and VAChT thus use leucine-based signals for efficient endocytosis and as such are the first synaptic vesicle proteins known to use this motif for trafficking.
Assuntos
Acetilcolina/metabolismo , Proteínas de Transporte/metabolismo , Endocitose , Leucina/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Neuropeptídeos , Neurotransmissores/metabolismo , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Animais , Células COS , Proteínas de Transporte/química , Proteínas de Transporte/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Homologia de Sequência de Aminoácidos , Proteínas Vesiculares de Transporte de Acetilcolina , Proteínas Vesiculares de Transporte de Aminas Biogênicas , Proteínas Vesiculares de Transporte de MonoaminaRESUMO
The vesicular monoamine transporters (VMATs) package monoamine neurotransmitters into secretory vesicles for regulated exocytotic release. One isoform occurs in the adrenal gland (VMAT1) and another in the brain (VMAT2). To assess their potential for regulation, we have investigated the phosphorylation of the VMATs. Using heterologous expression in Chinese hamster ovary, PC12, and COS cells, we find that rat VMAT2, but not VMAT1, is constitutively phosphorylated. Phosphoamino acid analysis indicates that this phosphorylation occurs on serine residues, and the analysis of VMAT1-VMAT2 chimeras and site-directed mutagenesis localize the phosphorylation sites to serines 512 and 514 at the carboxyl terminus of VMAT2. Since these residues occur in an acidic region, we tested the ability of the acidotropic kinases casein kinase I (CKI) and casein kinase II (CKII) to phosphorylate bacterial fusion proteins containing the carboxyl terminus of VMAT2. Purified CKI and CKII phosphorylate the wild-type carboxyl terminus of VMAT2, but not a double mutant with both serines 512 and 514 replaced by alanine. The protein kinase inhibitor CKI-7 and unlabeled GTP both block in vitro phosphorylation by cell homogenates, indicating a role for CKII and possibly CKI in vivo. Both kinases phosphorylate the VMAT2 fusion protein to a much greater extent than a similar fusion protein containing the carboxyl terminus of VMAT1, consistent with differential phosphorylation of the two transporters observed in intact cells. These results provide the first demonstration of phosphorylation of a vesicular neurotransmitter transporter and a potential mechanism for differential regulation of the two VMATs.