Equilibrative Nucleoside Transporter ENT1 as a Biomarker of Huntington Disease

The initial goal of this study was to investigate alterations in adenosine A2A receptor (A2AR) density or function in a rat model of Huntington disease (HD) with reported insensitivity to an A2AR antagonist. Unsuspected negative results led to the hypothesis of a low striatal adenosine tone and to the search for the mechanisms involved. Extracellular striatal concentrations of adenosine were measured with in vivo microdialysis in two rodent models of early neuropathological stages of HD disease, the Tg51 rat and the zQ175 knock-in mouse. In view of the crucial role of the equilibrative nucleoside transporter (ENT1) in determining extracellular content of adenosine, the binding properties of the ENT1 inhibitor [3H]-S-(4-Nitrobenzyl)-6-thioinosine were evaluated in zQ175 mice and the differential expression and differential coexpression patterns of the ENT1 gene (SLC29A1) were analyzed in a large human cohort of HD disease and controls. Extracellular striatal levels of adenosine were significantly lower in both animal models as compared with control littermates and striatal ENT1 binding sites were significantly upregulated in zQ175 mice. ENT1 transcript was significantly upregulated in HD disease patients at an early neuropathological severity stage, but not those with a higher severity stage, relative to non-demented controls. ENT1 transcript was differentially coexpressed (gained correlations) with several other genes in HD disease subjects compared to the control group. The present study demonstrates that ENT1 and adenosine constitute biomarkers of the initial stages of neurodegeneration in HD disease and also predicts that ENT1 could constitute a new therapeutic target to delay the progression of the disease

Cross-communication between Gi and Gs in a G-protein-coupled receptor heterotetramer guided by a receptor C-terminal domain

BACKGROUND: G-protein-coupled receptor (GPCR) heteromeric complexes have distinct properties from homomeric GPCRs, giving rise to new receptor functionalities. Adenosine receptors (A1R or A2AR) can form A1R-A2AR heteromers (A1-A2AHet), and their activation leads to canonical G-protein-dependent (adenylate cyclase mediated) and -independent (β-arrestin mediated) signaling. Adenosine has different affinities for A1R and A2AR, allowing the heteromeric receptor to detect its concentration by integrating the downstream Gi- and Gs-dependent signals. cAMP accumulation and β-arrestin recruitment assays have shown that, within the complex, activation of A2AR impedes signaling via A1R. RESULTS: We examined the mechanism by which A1-A2AHet integrates Gi- and Gs-dependent signals. A1R blockade by A2AR in the A1-A2AHet is not observed in the absence of A2AR activation by agonists, in the absence of the C-terminal domain of A2AR, or in the presence of synthetic peptides that disrupt the heteromer interface of A1-A2AHet, indicating that signaling mediated by A1R and A2AR is controlled by both Gi and Gs proteins. CONCLUSIONS: We identified a new mechanism of signal transduction that implies a cross-communication between Gi and Gs proteins guided by the C-terminal tail of the A2AR. This mechanism provides the molecular basis for the operation of the A1-A2AHet as an adenosine concentration-sensing device that modulates the signals originating at both A1R and A2AR

Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer

Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain

Identification and characterization of Adenosine A(2A) heteromers in the CNS = Identificació i caracterització d’heteròmers d’Adenosina A2A al SNC

[spa] Els objectius de la tesi doctoral són: - Objectiu 1. Implicacions de l’heteròmer A(1)R-A(2A)R a les cèl•lules glials i el seu estudi a nivell molecular. - Objectiu 2. Trobar evidències de modulacions al•lostèriques entre receptors a l’heteròmer A(2A)DD(2)R que confereixen característiques farmacològiques específiques a l’heteròmer. - Objectiu 3. Recerca d’antagonistes selectius de A(2A)R per a heteròmers presinàptics A(1)R-A(2A)R versus heteròmers postsinàptics A(2A)R-D(2)R que poden ser útils en el tractament de malalties neurològiques, particularment en la malaltia de Huntington. - Objectiu 4. Investigar les propietats funcionals i farmacològiques de A(2A)R en l’heteròmer A(2A)RCB(1)R. - Objectiu 5. Anàlisi de diferents compostos antagonistes de A(2A)R en línies cel•lulars estables CHO expressant A(2A)R, A(1)R-A(2A)R, A(2A)R-D(2)R o A(2A)R-CB(1)R. Les conclusions de la tesi són les següents. Primer objectiu: - En la recaptació de GABA, l’adenosina te un efecte bifàsic, el qual està mediat pels heteròmers A(1)R-A(2A)R que es troben acoblats a proteïnes Gi/o i Gs. L’adenosina extracel•lular actuant sobre aquests heteròmers opera en el balanç de recaptació de GABA depenent de l’activitat PKA. La senyalització neural serà inhibitòria a baixa activació neuronal i facilitadora a alta activitat neuronal. - BRET i experiments de “single molecule tracking” amb microscopi TIRF demostren que l’expressió mínima d’aquest heteròmer consta de quatre protomers i dues proteïnes G. La gran similitud entre GPCR suggereix que aquest model molecular podria ser aplicable a altres receptors. - Aquests heteròmers poden formar-se a la membrana plasmàtica i són estables durant minuts. Aquesta estabilitat suggereix que el disseny de fàrmacs dirigits contra aquests heteròmers és una estratègia viable. Segon objectiu: - En cultius cel•lulars, la unió d’agonistes i antagonistes a A(2A)R fa disminuir l’afinitat d’agonistes i antagonistes per al D(2)R. - Aquestes interaccions negatives entre lligands són conseqüència d’interaccions al•lostèriques entre ambdós receptors que conformen l’heteròmer A(2A)R-D(2)R i constitueixen una unitat bioquímica. - El fet de que els antagonistes de A(2A)R són capaços de modular la farmacologia de D(2)R ha de ser tingut en compte per poder entendre patologies com la malaltia de Parkinson i per a la neuroimatge per PET. Tercer objectiu: - La presència física de D(2)R en l’heteròmer A(2A)R-D(2)R indueix una forta cooperativitat negativa a A(2A)R la qual va ser detectada per SCH 442416. Aquesta cooperativitat indica que el homodimers A(2A)R-A(2A)R es troben presents a l’heteròmer A(2A)R-D(2)R. - Basant-nos en experiments in vitro i in vivo, el compost SCH 442416 va ser classificat com a preferentment antagonista de receptor A(2A) presinàptic, i el compost KW 6002 va ser classificat com a antagonista preferentment postsinàptic. Considerant això, SCH 442416 pot ser utilitzat pel desenvolupament de fàrmacs antidiscinètics pel tractament de la malaltia de Huntington, mentre que KW 6002 pot ser beneficiós per a tractar la malaltia de Parkinson. Quart objectiu: - A(2A)R canvia el seu acoblament a proteïna Gs per Gi quan passa a formar heteròmers amb CB(1)R i un “cross-talk” sinergístic en activació de proteïna G s’observa quan tots dos receptors es troben co-activats. Cinquè objectiu: - El compost número nou podria ser un bon candidat per tractar la malaltia de Parkinson degut a la seva unió preferencial al receptor A(2A) present a l’heteròmer A(2A)R-D(2)R.[eng] The aims of this thesis are: Aim 1. The involvement of A(1)R-A(2A)R heteromer in glial cells and its study at a molecular level. Aim 2. To find evidence for allosteric interactions between partner receptors in the A(2A)RD(2)R receptor heteromer which confer specific pharmacological characteristics to the heteromer. Aim 3. Search for selective antagonists of A(2A)R for presynaptic A(1)R-A(2A)R heteromers versus postsynaptic A(2A)R-D(2)R heteromers that can be useful for treatment of neurological disorder’s treatment, particularly Huntington’s disease. Aim 4. Investigate the pharmacological and functional properties of A(2A)R in the A(2A)CB1 heteromer. Aim 5. Compound screening of different A(2A)R antagonists in stable CHO cell lines expressing A(2A)R, A(1)R-A(2A)R, A(2A)R-D(2)R or A(2A)R-CB(1)R heteromers. The conclusions of the objectives are: Conclusions derived from the first aim: The involvement of A(1)R-A(2A)R heteromer in glial cells and its study at a molecular level. - Upon GABA uptake, adenosine has a biphasic effect, which is mediated by A(1)RA(2A)R heteromers coupled to both Gi/0 and Gs proteins. Extracellular adenosine acting on these A(1)R-A(2A)R functional units operates in a concerted way to balance a PKA-dependent action on GABA uptake. The neural output would thus be inhibitory at low firing rates and facilitatory at high firing rates. - Adenosine by acting on adenosine receptors in astrocytes may significantly contribute to neurotransmission in a dual manner, which depends on the concentration of the nucleoside that is in turn dependent on neuronal firing activity. - BRET and single molecule tracking with TIRF microscope show that the minimal GPCR heteromer unit may consist of four protomers and two G proteins. The strong similarity between GPCRs suggests that the molecular model proposed could apply to other receptors. - These heteromers can be formed in the plasma membrane and are stable on the order of minutes. Such stability suggests that designing ways to target these heteromers may indeed be a viable strategy. - The orientation of the alpha-subunits of the G proteins is on the distal receptors, suggesting that G proteins cross-talk could occur via receptors across the heteromer complex. - The heteromeric unit described, with its dynamic and structural limitations, provides the molecular framework to understand why heteromers are functionally distinct units and not merely the aggregation of two entities with independent functions. Conclusions derived from the second aim: To find an evidence for allosteric interactions between partner receptors in the A(2A)R-D(2)R receptor heteromer which confer specific pharmacological characteristics to the heteromer. In cell culture, the agonist and antagonist binding to the adenosine A(2A)R diminish the affinity of dopamine D(2)R agonists and antagonists. - Those negative interactions between ligands are consequence of allosteric interactions between both receptors conforming the A(2A)R-D(2)R heteromer and constitute a unique biochemical property of this heteromer. - In ex vivo tissue, using these allosteric interactions as a heteromer fingerprint, it has been demonstrated the expression of A(2A)R-D(2)R heteromer in human striatum. - The fact that the A(2A)R antagonists are able to modulate dopamine D(2)R pharmacology has to be taken into account to understand pathologies such as Parkinson’s disease or for human PET neuroimaging. Conclusions derived from the third aim: Search for selective antagonists of A(2A)R for presynaptic A(1)R-A(2A)R heteromers versus postsynaptic A(2A)R-D(2)R heteromers that can be useful for neurological disorder’s treatment, particularly Huntington’s disease. - The physical presence of dopamine D(2)R in the A(2A)R-D(2)R heteromer induced a strong negative cooperativity in the A(2A)R that was detected by SCH-442416. This cooperativity indicates that A(2A)R-A(2A)R homodimers are present in the A(2A)R-D(2)R heteromer. - Based on in vitro and in vivo approaches, the compound SCH-442416 was classified as a preferential presynaptic A(2A)R antagonist, and the compound KW- 6002 was classified as a preferential postsynaptic A(2A)R antagonist. Considering this, SCH-442416 can be used as a lead compound in the development of antidyskinetic drugs in Huntington’s disease; meanwhile KW-6002 can be beneficial in Parkinson’s disease. Conclusions derived from the fourth aim: Investigate the pharmacological and functional properties of A(2A)R in the A(2A)R-CB1R heteromer. - Adenosine A(2A)R changes its G-protein coupling from stimulatory Gs to inhibitory Gi when it forms heteromer with CB1R and a synergistic cross-talk in G-protein activation is observed when both receptors are coactivated. - CB1R mainly controls the ERK1/2 signaling under the A(2A)R-CB1R heteromer. - The A(2A)R-CB1R heteromer does not show allosteric effects at the ligand binding level. Conclusions derived from the fifth aim: Compound screening of different A(2A)R antagonists in stable CHO cell lines expressing A(2A)R, A(1)R-A(2A)R, A(2A)R-D(2)R or A(2A)R-CB1R heteromers. - Compound number 9 could be a good candidate to treat Parkinson’s disease due to its preferential binding to A(2A)R forming A(2A)R-D(2)R heteromer

Equilibrative Nucleoside Transporter ENT1 as a Biomarker of Huntington Disease

The initial goal of this study was to investigate alterations in adenosine A2A receptor (A2AR) density or function in a rat model of Huntington disease (HD) with reported insensitivity to an A2AR antagonist. Unsuspected negative results led to the hypothesis of a low striatal adenosine tone and to the search for the mechanisms involved. Extracellular striatal concentrations of adenosine were measured with in vivo microdialysis in two rodent models of early neuropathological stages of HD disease, the Tg51 rat and the zQ175 knock-in mouse. In view of the crucial role of the equilibrative nucleoside transporter (ENT1) in determining extracellular content of adenosine, the binding properties of the ENT1 inhibitor [3H]-S-(4-Nitrobenzyl)-6-thioinosine were evaluated in zQ175 mice and the differential expression and differential coexpression patterns of the ENT1 gene (SLC29A1) were analyzed in a large human cohort of HD disease and controls. Extracellular striatal levels of adenosine were significantly lower in both animal models as compared with control littermates and striatal ENT1 binding sites were significantly upregulated in zQ175 mice. ENT1 transcript was significantly upregulated in HD disease patients at an early neuropathological severity stage, but not those with a higher severity stage, relative to non-demented controls. ENT1 transcript was differentially coexpressed (gained correlations) with several other genes in HD disease subjects compared to the control group. The present study demonstrates that ENT1 and adenosine constitute biomarkers of the initial stages of neurodegeneration in HD disease and also predicts that ENT1 could constitute a new therapeutic target to delay the progression of the disease

Striatal pre- and postsynaptic profile of adenosine A2A receptor antagonists

Striatal adenosine A2A receptors (A2ARs) are highly expressed in medium spiny neurons (MSNs) of the indirect efferent pathway, where they heteromerize with dopamine D2 receptors (D2Rs). A2ARs are also localized presynaptically in cortico-striatal glutamatergic terminals contacting MSNs of the direct efferent pathway, where they heteromerize with adenosine A1 receptors (A1Rs). It has been hypothesized that postsynaptic A2AR antagonists should be useful in Parkinson's disease, while presynaptic A2AR antagonists could be beneficial in dyskinetic disorders, such as Huntington's disease, obsessive-compulsive disorders and drug addiction. The aim or this work was to determine whether selective A2AR antagonists may be subdivided according to a preferential pre- versus postsynaptic mechanism of action. The potency at blocking the motor output and striatal glutamate release induced by cortical electrical stimulation and the potency at inducing locomotor activation were used as in vivo measures of pre- and postsynaptic activities, respectively. SCH-442416 and KW-6002 showed a significant preferential pre- and postsynaptic profile, respectively, while the other tested compounds (MSX-2, SCH-420814, ZM-241385 and SCH-58261) showed no clear preference. Radioligand-binding experiments were performed in cells expressing A2AR-D2R and A1R-A2AR heteromers to determine possible differences in the affinity of these compounds for different A2AR heteromers. Heteromerization played a key role in the presynaptic profile of SCH-442416, since it bound with much less affinity to A2AR when co-expressed with D2R than with A1R. KW-6002 showed the best relative affinity for A2AR co-expressed with D2R than co-expressed with A1R, which can at least partially explain the postsynaptic profile of this compound. Also, the in vitro pharmacological profile of MSX-2, SCH-420814, ZM-241385 and SCH-58261 was is in accordance with their mixed pre- and postsynaptic profile. On the basis of their preferential pre- versus postsynaptic actions, SCH-442416 and KW-6002 may be used as lead compounds to obtain more effective antidyskinetic and antiparkinsonian compounds, respectively

Cocaine inhibits dopamine D2 receptor signaling via sigma-1-D2 receptor heteromers

Under normal conditions the brain maintains a delicate balance between inputs of reward seeking controlled by neurons containing the D1-like family of dopamine receptors and inputs of aversion coming from neurons containing the D2-like family of dopamine receptors. Cocaine is able to subvert these balanced inputs by altering the cell signaling of these two pathways such that D1 reward seeking pathway dominates. Here, we provide an explanation at the cellular and biochemical level how cocaine may achieve this. Exploring the effect of cocaine on dopamine D2 receptors function, we present evidence of σ1 receptor molecular and functional interaction with dopamine D2 receptors. Using biophysical, biochemical, and cell biology approaches, we discovered that D2 receptors (the long isoform of the D2 receptor) can complex with σ1 receptors, a result that is specific to D2 receptors, as D3 and D4 receptors did not form heteromers. We demonstrate that the σ1-D2 receptor heteromers consist of higher order oligomers, are found in mouse striatum and that cocaine, by binding to σ1 -D2 receptor heteromers, inhibits downstream signaling in both cultured cells and in mouse striatum. In contrast, in striatum from σ1 knockout animals these complexes are not found and this inhibition is not seen. Taken together, these data illuminate the mechanism by which the initial exposure to cocaine can inhibit signaling via D2 receptor containing neurons, destabilizing the delicate signaling balance influencing drug seeking that emanates from the D1 and D2 receptor containing neurons in the brain

Cocaine inhibits dopamine D2 receptor signaling via sigma-1-D2 receptor heteromers

Under normal conditions the brain maintains a delicate balance between inputs of reward seeking controlled by neurons containing the D1-like family of dopamine receptors and inputs of aversion coming from neurons containing the D2-like family of dopamine receptors. Cocaine is able to subvert these balanced inputs by altering the cell signaling of these two pathways such that D1 reward seeking pathway dominates. Here, we provide an explanation at the cellular and biochemical level how cocaine may achieve this. Exploring the effect of cocaine on dopamine D2 receptors function, we present evidence of σ1 receptor molecular and functional interaction with dopamine D2 receptors. Using biophysical, biochemical, and cell biology approaches, we discovered that D2 receptors (the long isoform of the D2 receptor) can complex with σ1 receptors, a result that is specific to D2 receptors, as D3 and D4 receptors did not form heteromers. We demonstrate that the σ1-D2 receptor heteromers consist of higher order oligomers, are found in mouse striatum and that cocaine, by binding to σ1 -D2 receptor heteromers, inhibits downstream signaling in both cultured cells and in mouse striatum. In contrast, in striatum from σ1 knockout animals these complexes are not found and this inhibition is not seen. Taken together, these data illuminate the mechanism by which the initial exposure to cocaine can inhibit signaling via D2 receptor containing neurons, destabilizing the delicate signaling balance influencing drug seeking that emanates from the D1 and D2 receptor containing neurons in the brain

Cross-communication between Gi and Gs in a G-protein-coupled receptor heterotetramer guided by a receptor C-terminal domain

BACKGROUND: G-protein-coupled receptor (GPCR) heteromeric complexes have distinct properties from homomeric GPCRs, giving rise to new receptor functionalities. Adenosine receptors (A1R or A2AR) can form A1R-A2AR heteromers (A1-A2AHet), and their activation leads to canonical G-protein-dependent (adenylate cyclase mediated) and -independent (β-arrestin mediated) signaling. Adenosine has different affinities for A1R and A2AR, allowing the heteromeric receptor to detect its concentration by integrating the downstream Gi- and Gs-dependent signals. cAMP accumulation and β-arrestin recruitment assays have shown that, within the complex, activation of A2AR impedes signaling via A1R. RESULTS: We examined the mechanism by which A1-A2AHet integrates Gi- and Gs-dependent signals. A1R blockade by A2AR in the A1-A2AHet is not observed in the absence of A2AR activation by agonists, in the absence of the C-terminal domain of A2AR, or in the presence of synthetic peptides that disrupt the heteromer interface of A1-A2AHet, indicating that signaling mediated by A1R and A2AR is controlled by both Gi and Gs proteins. CONCLUSIONS: We identified a new mechanism of signal transduction that implies a cross-communication between Gi and Gs proteins guided by the C-terminal tail of the A2AR. This mechanism provides the molecular basis for the operation of the A1-A2AHet as an adenosine concentration-sensing device that modulates the signals originating at both A1R and A2AR

Quaternary structure of a G-protein-coupled receptor heterotetramer in complex with Gi and Gs

Background G-protein-coupled receptors (GPCRs), in the form of monomers or homodimers that bind heterotrimeric G proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. Different GPCRs may also interact to form heteromers that are novel signaling units. Despite the exponential growth in the number of solved GPCR crystal structures, the structural properties of heteromers remain unknown. Results We used single-particle tracking experiments in cells expressing functional adenosine A1-A2A receptors fused to fluorescent proteins to show the loss of Brownian movement of the A1 receptor in the presence of the A2A receptor, and a preponderance of cell surface 2:2 receptor heteromers (dimer of dimers). Using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and G-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the GPCR tetramer in complex with two G proteins. Conclusions The combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric G proteins (Gi and Gs). These novel results constitute an important advance in understanding the molecular intricacies involved in GPCR function