Localisation subcellulaire des deux isoformes du récepteur D2 de la dopamine chez les mamifères (approche "in vitro")

Chez les mammifères, le récepteur D2 existe sous forme de deux variants moléculaires issu d'un épissage alternatif de son ARN prémessager, le récepteur D-2s (isoforme courte) et le récepteur D-2L (isoforme longue), qui diffèrent de 29 acides aminés au sein de la troisième boucle cytoplasmique. Les conséquences fonctionnelles de cet épissage sont cependant encore très mal connues. La localisation subcellulaire des récepteurs étant un des paramètres essentiels de leur fonction, nous avons donc cherché à étudier l'existence d'une localisation et d'une régulation intracellulaire différentielle des isoformes D-2s et D-2L. Le résultat principal de cette étude est l'observation d'une localisation prédominante des récepteurs D-2s et D-2L dans des compartiments intracellulaires, identifiés comme étant principalement le réticulum endoplasmique et pour une moindre part l'appareil de Golgi, sans différence significative entre les deux isoformes du récepteur D2. Nous avons montré que ce phénomène d'accumulation intracellulaire s'accompagne d'une vacuolisation massive de la morphologie du réticulum endoplasmique. La mise en évidence d'une activité intrinsèque importante des récepteurs D-2s et D-2L semble être responsable, au moins en partie, de cette perturbation. Dans les cultures primaires de striatum de rat, nous avons pu montrer que les récepteurs D2 sont localisés, pour une part importante, dans des membranes intracellulaires des neurones, système proche des conditions naturelles. L'analyse conjointe de l'effet de traitement des récepteurs D2 par différents types de ligands pharmacologiques n'a révélé aucun changement de localisation membranaire ou intracellulaire des isoformes que ce soit dans les cellules hétérologues ou les neurones de striatum en culture primaire. L'ensemble de ces résultats semblent indiquer que les récepteurs D2 ont une localisation subcellulaire et un fonctionnement atypique par rapport à ce que l'on connaît aujourd'hui de la biologie des récepteurs couplés aux protéines G.In mammals, the dopamine D2 receptor exists as a long (D2a) and a short (D2b) isoforms generated by alternative splicing of the corresponding transcript, which modifies the length of the third cytoplasmic loop implicated in heterotrimeric G protein-coupling. The functional consequences of this splicing event are still unclear but it may be involved in the mechanisms of the intracellular transport of the protein. To directly address this question, we used a combination of tagging procedures and immunocytochemical techniques to detect each of the two D2 receptor isoforms. The tagged receptor functionally behaving as wild type receptors, we performed transient expression in non neuronal and neuronal cell lines, and in rat striatum primary cultures. Surprisingly, in transfected heterologous cells, most of the newly synthesized receptors accumulate in large intracellular compartments, the plasma membrane being only weakly labeled, without significant difference between the two receptor isoforms. Double labeling experiments showed the D2 receptor is mostly retained in the endoplasmic reticulum (ER) and in the Golgi apparatus for a fewer part. This retention is accompanied by a striking vacuolization of the ER. This phenomenon, as well as the intracellular retention of the D2 receptors, is partly due to an intrinsic activity of the D2 receptor isoforms. In rat striatum primary culture, the receptors are more localized at the plasma membrane than in the heterologous cell lines, but a significant retention of the receptors is still observed in intracellular compartments. The treatment of the cells with different D2 receptor ligands did not trigger any change in the D2 receptors localization, either in transfected cell lines or in neurons. These results show that the D2 dopamine receptors, as compared to others G protein coupled receptors, have a peculiar subcellular localization and seem to have unusual regulatory mechanisms.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Autocrine activation of adenosine A1 receptors blocks D1A but not D1B dopamine receptor desensitization.

Adenosine is known to modulate dopamine responses in several brain areas. Here, we show that tonic activation of adenosine receptors is able to impede desensitization of D1 dopamine receptors. As measured by cAMP accumulation in transfected COS-7 cells, long-term exposure to dopamine agonists promoted desensitization of D1B receptor but not that of D1A receptor. The inability of D1A receptor to desensitize was a result of the adenosine present in culture medium acting through activation of adenosine A1 receptors. Cell incubation with either adenosine deaminase, CGS-15943, a generic adenosine receptor antagonist, or the A1 antagonist DPCPX restored the long-term desensitization time-course of D1A receptors. In Ltk cells stably expressing A1 adenosine receptors and D1A dopamine receptors, pre-treatment of cells with R(-)-PIA, a full A1 receptor agonist, did not significantly inhibit the acute increase in cAMP levels induced by D1 receptor agonists, but blocked desensitization of D1A receptors. However, simultaneous activation of A1 and D1A receptors promoted a delayed D1A receptor desensitization. This suggests that functional interaction between A1 and D1A receptors may depend on the activation kinetics of components regulating D1 receptor responses, acting differentially on D1A and D1B receptors

Modulation of dopamine transporter function by alpha-synuclein is altered by impairment of cell adhesion and by induction of oxidative stress.

Human alpha-synuclein accumulates in dopaminergic neurons as intraneuronal inclusions, Lewy bodies, which are characteristic of idiopathic Parkinson's disease (PD). Here, we suggest that modulation of the functional activity of the dopamine transporter (DAT) by alpha-synuclein may be a key factor in the preferential degeneration of mesencephalic dopamine (DA)-synthesizing neurons in PD. In cotransfected Ltk-, HEK 293, and SK-N-MC cells, alpha-synuclein induced a 35% decrease in [3H]DA uptake. Biotinylated DAT levels were decreased by 40% in cotransfected cells relative to cells expressing only DAT. DAT was colocalized with alpha-synuclein in mesencephalic neurons and cotransfected Ltk- cells. Coimmunoprecipitation studies showed the existence of a complex between alpha-synuclein and DAT, in specific rat brain regions and cotransfected cells, through specific amino acid motifs of both proteins. The attenuation of DAT function by alpha-synuclein was cytoprotective, because DA-mediated oxidative stress and cell death were reduced in cotransfected cells. The neurotoxin MPP+ (1-methyl-4-phenylpyridinium), oxidative stress, or impairment of cell adhesion ablated the alpha-synuclein-mediated inhibition of DAT activity, which caused increased uptake of DA and increased biotinylated DAT levels, in both mesencephalic neurons and cotransfected cells. These studies suggest a novel normative role for alpha-synuclein in regulating DA synaptic availability and homeostasis, which is relevant to the pathophysiology of PD

Mutations in the lipid-binding domain of alpha-synuclein confer overlapping, yet distinct, functional properties in the regulation of dopamine transporter activity.

Alpha-synuclein and its missense mutants (A30P, A53T) have been linked to the genesis of idiopathic and rare familial forms of Parkinson's disease, respectively. Here we show that, similar to the wild-type alpha-synuclein, the A30P mutant forms a strong complex with the human dopamine transporter (hDAT), through direct protein:protein interactions between the nonamyloid beta component (NAC) domain of the A30P mutant and the last 22 aminoacyl residues of the carboxy-terminal tail of hDAT. The A30P mutant negatively modulates hDAT functional activity and to a greater extent than wild-type alpha-synuclein, with reduced uptake of extracellular dopamine and dopamine-mediated, hDAT-dependent cytotoxicity. By contrast, the A53T mutant neither forms a strong protein:protein complex with hDAT nor modulates dopamine uptake by hDAT, and dopamine-mediated, hDAT-dependent cytotoxicity is higher than with either wild-type or the A30P variant of alpha-synuclein, but not significantly different from that of cells expressing hDAT alone. Confocal microscopy shows substantial overlap in colocalization of all three alpha-synuclein variants with hDAT, with only minor differences. Although the complex formation with hDAT occurs through the NAC domain of the alpha-synuclein variants, it is the familial Parkinson's disease-linked missense mutations present in the amino-terminal lipid binding domain of the alpha-synuclein variants that dictate the extent of the regulation of hDAT function. These studies highlight previously unknown properties of the A30P and the A53T mutants of alpha-synuclein with respect to the modulation of hDAT activity and/or regulation, and its subsequent functional outcome, which are uniquely distinct

Oral noribogaine shows high brain uptake and anti-withdrawal effects not associated with place preference in rodents

This study investigated the effects of noribogaine, the principal metabolite of the drug ibogaine, on substance-related disorders. In the first experiment, mice chronically treated with morphine were subjected to naloxone-precipitated withdrawal two hours after oral administration of noribogaine. Oral noribogaine dose dependently decreased the global opiate withdrawal score by up to 88% of vehicle control with an ED50 of 13 mg/kg. In the second experiment, blood and brain levels of noribogaine showed a high brain penetration and a brain/blood ratio of 7±1 across all doses tested. In a third experiment, rats given oral noribogaine up to 100 mg/kg were tested for abuse liability using a standard biased conditioned place paradigm. Noribogaine-treated rats did not display place preference, suggesting that noribogaine is not perceived as a hedonic stimulus in rodents. Retrospective review of published studies assessing the efficacy of ibogaine on morphine withdrawal shows that the most likely cause of the discrepancies in the literature is the different routes of administration and time of testing following ibogaine administration. These results suggest that the metabolite noribogaine rather than the parent compound mediates the effects of ibogaine on blocking naloxone-precipitated withdrawal. Noribogaine may hold promise as a non-addicting alternative to standard opiate replacement therapies to transition patients to opiate abstinence

Intracellular retention of the two isoforms of the D(2) dopamine receptor promotes endoplasmic reticulum disruption.

The dopamine D(2) receptor exists as a long (D(2a)) and a short (D(2b)) isoform generated by alternative splicing of the corresponding transcript, which modifies the length of the third cytoplasmic loop implicated in heterotrimeric G-protein-coupling. Anatomical data suggested that this segment regulates the intracellular traffic and localization of the receptor. To directly address this question we used a combination of tagging procedures and immunocytochemical techniques to detect each of the two D(2) receptor isoforms. Surprisingly, most of the newly synthesized receptors accumulate in large intracellular compartments, the plasma membrane being only weakly labeled, without significant difference between the two receptor isoforms. Double labeling experiments showed that this localization corresponded neither to endosomal compartments nor to the Golgi apparatus. The D(2) receptor is mostly retained in the endoplasmic reticulum (ER), the long isoform more efficiently than the short one. It is accompanied by a striking vacuolization of the ER, roughly proportional to the expression levels of the two receptor isoforms. This phenomenon is partly overcome by treatment with pertussis toxin. In addition, an intrinsic activity of the D(2) receptor isoforms is revealed by [(35)S]-GTP gamma S binding and cAMP assay, which suggested that expression of weakly but constitutively active D(2) receptors promotes activation of heterotrimeric G protein inside the secretory pathway. This mechanism may participate in the regulation of the cellular traffic of the D(2) receptors isoforms

Evolution and cell biology of dopamine receptors in vertebrates.

Dopamine, one of main modulatory neurotransmitters of the nervous system acts on target cells through two classes of G protein-coupled receptors, D1 and D2. The two dopamine receptor classes display different structures, interact with different regulatory partners (including heterotrimeric G proteins) and, accordingly, have independent evolutionary origins. In vertebrates, each of these receptor classes comprises several subtypes, generated by two steps of gene duplications, early in vertebrate evolution. In the D1 receptor class, the D1A, D1B, D1C and D1D subtypes, and in the D2 class, the D2, D3 and D4 receptor subtypes have been conserved in most vertebrate groups. This conservation has been driven by the acquisition, by each receptor subtype, of a small number of specific properties, which were selected for adaptive purpose in vertebrates. Among these properties, affinity for dopamine, the natural ligand, intrinsic receptor activity, and agonist-induced desensitization clearly distinguish the receptor subtypes. In addition, each dopamine receptor subtype is addressed to a specific location within neuronal networks, although detailed information is lacking for several receptor subtypes. Receptors localization at diverse subcellular places in neurons may also differ from one subtype to another, resulting in different ways of regulating cell signalisation. One challenge for future research on dopamine and its receptors would be to identify the nature of the protein partners and the molecular mechanisms involved in localizing receptors to the neuronal plasma membrane. In this respect, the evolutionary approach we have undertaken suggests that, due to gene duplications, a reasonable degree of freedom exists in the tight organisation of dopamine receptors in neurons. This "evolvability" of dopamine systems has been instrumental to adapt the vertebrate species to nearly all the possible environments