GPR37-ADENOSINE A2A receptor-receptor interaction as a new pharmacological target for Parkinson’s disease treatment
GPR37 is an orphan GPCR that belongs to the Rodhopsin family. GPR37 is highly expressed in the Central Nervous System (CNS), while little peripheral expression has been found in humans and rodents. GPR37 is highly distributed in the brain, particularly at the cerebellum, corpus callosum, substantia...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2017 |
| País: | España |
| Institución: | CBUC, CESCA |
| Repositorio: | TDR. Tesis Doctorales en Red |
| OAI Identifier: | oai:www.tdx.cat:10803/457874 |
| Acceso en línea: | http://hdl.handle.net/10803/457874 |
| Access Level: | acceso abierto |
| Palabra clave: | Malaltia de Parkinson Enfermedad de Parkinson Parkinson's disease Marcadors bioquímics Marcadores bioquímicos Biochemical markers Ciències de la Salut 615 |
| Sumario: | GPR37 is an orphan GPCR that belongs to the Rodhopsin family. GPR37 is highly expressed in the Central Nervous System (CNS), while little peripheral expression has been found in humans and rodents. GPR37 is highly distributed in the brain, particularly at the cerebellum, corpus callosum, substantia nigra, striatum and hippocampus with a predominantly neuronal distribution. However, the function of this receptor in the CNS remains unknown. The interest on GPR37 biology was bolstered when it was described as a Parkin substrate. GPR37 was isolated from a human brain library in a yeast 2-hybrid screen for novel interacting partners of Parkin, and thus rebaptized as “Parkin-associated endothelin-like receptor” (Pael-R). Parkin is of great interest because mutations in this gene are directly linked to autosomal recessive juvenile Parkinsonism (AR-JP). It was found that insoluble GPR37 was increased in patients with AR-JP, linking the aggregation of GPR37 to disease pathogenesis. Later, GPR37 aggregates were also found in a variety of inclusion bodies in brains from patients with PD, dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). All these data suggested a relevant role of the GPR37 in the aggregates formation in the PD. Our working hypothesis is based on that this GPR37 oligomerization with the adenosine and dopamine receptors, together with the neuropathological changes during PD can be relevant during PD development and basal ganglia function, and thus for the PD ethiopatology. Finally, in addition to elucidate GPR37 function in the CNS, in this doctoral thesis we have studied different specific ligand candidates for this orphan receptor. RESULTS Elucidating the existence and functional consequences of a direct GPR37-A2AR receptor- receptor interaction in the striatum. Two polyclonal antibodies which recognized the extracellular N-terminal or the intracellular C-terminal fragment of GPR37 respectively were produced by the immunization of rabbits (strain New Zealand White) in the Animal house facilities in the Bellvitge Health Sciences Campus. The results obtained by Western Blot using the antibody against the N-terminal region of the GPR37 receptor was a single specific band of about 53 kDa, similar to the theoretical weight calculated for the receptor peptidic chain. On the other hand, the result obtained by Western Blot using the antibody against the C-terminal region of the GPR37 receptor was a single specific band of about 40 kDa, an unexpected result since the expected size of the complete GPR37 receptor is approximately 53 kDa. Our results confirmed this N-terminal cleavage of the receptor in native tissue. In our work, we studied with special emphasis the physical and functional interaction between the GPR37 and A2AR receptor in native tissue, specifically in striatum. First, we verified the presence, co-distribution and direct interaction of GPR37 and A2AR by different methods: i) Through histoblot and immunohistochemistry (IHC) we confirmed that the two receptors co-distribute in the striatum. ii) Next, by means of electron microscopy and the technique of subsynaptic fractionation we characterized the subsynaptic distribution of these receptors in the striatum, observing that both receptors were enriched at the postsynaptic level. iii) Using co-immunoprecipitation (Co-ip), the proximity ligation assay (PLA) technique and double labeling with gold immunoparticles (in collaboration with Prof. Rafael Luján, UCLM, Albacete), we demonstrated the direct interaction between both receptors in the striatum. Subsequently, we analyzed the impact of the presence/ absence of GPR37 on the expression and functionality of A2AR. Interestingly, GPR37 deletion promoted an increase in A2AR membrane expression in the striatum observed by biotinylation of membrane proteins performed on corticostriatal slices. On the other hand, we determined the generation of A2AR-mediated cAMP after stimulation with CGS21680, a selective agonist of this receptor, in total synaptosomes and primary cultures of striatum. Interestingly, the results showed a greater effect of the A2AR agonist on GPR37-/- mice when compared with the Gpr37+/+ mice. In addition, we performed behavioral tests to analyze striatal-dependent functions (i.e. locomotor activity) to assess the relevance of GPR37 receptor and its impact on A2AR function by administering SCH58261 (3.75mg/kg, intraperitoneal, i.p.), a selective A2AR receptor, in GPR37+/+ and GPR37-/- mice. In the Open field test, mice freely explored the field. The horizontal locomotor activity (total distance traveled) was analyzed and a greater increase of the spontaneous locomotor activity in the GPR37-/- animals treated with SCH58261 was observed. Finally, we characterize the role of GPR37 in an animal model of PD, and its relation with the adenosinergic transmission. PD is characterized by a degeneration of the nigrostriatal dopaminergic neurons, resulting in an imbalance of the function of the basal ganglia. Pharmacological treatment of PD is mainly based on improving dopaminergic transmission (either by enhancing the release of dopamine or by directly activating dopamine receptors). However, severe side effects appear after prolonged treatment, including dyskinesias and somnolence. During the last decades, the A2AR antagonists have been studied as a good alternative or supplementation (together with the classical dopaminergic drugs) for the treatment of PD. A2AR antagonists mechanism, is based on the interaction between these receptors and the D2R in the striatum. For example, A2AR antagonists attenuate catalepsy induced by D2R blockade in rodents. In addition, this catalytic effect can also be induced by the administration of A2AR agonists at high doses. We used the pharmacological catalepsy model to evaluate the impact of the presence of GPR37 on the adenosinergic transmission in the striatum. First, we administered haloperidol, a D2R antagonist, together with SCH58261, and quantified the duration of the cataleptic effect. Next, we used the CGS21680, a selective A2AR agonist, administered intracerebroventricularly (i.c.v.), to cause the cataleptic effect. The results showed an increase in the cataleptic response in GPR37-/- animals which would correlate with an increase in the A2AR expression in the synapse previously observed. In order to study the role of this orphan receptor in CNS synaptic plasticity and to evaluate its involvement in A2AR receptor function, we performed behavioral experiments related to striatal functions in GPR37+/+ and GPR37-/- mice of 2 months old mice administered chronically with SCH58261 (1mg/kg, i.p. for 14 days). Thus, we performed the following behavioral tests: i) Open field test and ii) Modified Water maze test. These tests allowed us to evaluate locomotor activity and learning memory, respectively. Subsequently, extracellular field electrophysiological recordings were performed on corticostriatal slices to evaluate possible changes in the synaptic activity and plasticity of these animals, and their relationship with GPR37 receptor or chronic treatment with the A2AR antagonist. In conclusion, the results obtained in the behavioral experiments and LTD electrophysiological records performed in corticostriatal slices, made us hypothesize that in the absence of the GPR37 receptor, sensitization to A2AR receptor antagonists occurs, due to differences in the levels of this receptor in the membrane. This would coincide with our results previously obtained in the laboratory, where using techniques such as biotinylation of membrane receptors or subsynaptic fractionation, where we can quantify the amount of receptors in pre- and postsynaptic densities, we observed an increase in A2AR receptor expression in the plasma membrane in the Gpr37 - / - animals. Establishing the existence of GPR37-A2AR in other brain areas, such as the hippocampus, describing the synaptic distribution of GPR37 and determining its role in A2AR-mediated synaptic plasticity. The results obtained with this work described the presence of this orphan receptor in the hippocampus and a preferably postsynaptic location in the neurons. In addition, a series of behavioral tests related to hippocampal dependent functions (i.e. anxiety and memory) were also performed to evaluate the relevance of the GPR37. We also evaluated the interaction of GPR37 with the A2AR by administering SCH58261. Novel Object Recognition (NOR) test, widely used to study effects on memory. On the other hand the Marble Burying Test (MBT) and the Elevated Plus Maze (EPM) were performed to evaluate the anxiety. The results obtained coincide with previous studies, which showed that the deletion of A2AR has an anxiogenic effect and that overexpression leaded to a less anxious phenotype. Evaluating GPR37 receptor expression levels in PD and the potential use of this receptor as a PD biomarker. The expression/ recycling of GPR37 in some types of PD is altered, triggering an increase and accumulation of this receptor in neurons. In this objective, we intend to describe whether this process occurs only in an isolated group of PD patients (AR-JP, a genetic cause) or if it was a more widespread process in PD, thus being able to describe its histopathological evolution through the different stages of the disease. In addition, this increase of the GPR37 receptor in the tissues of the CNS could translate into an increase in the presence of the receptor or fragments of this receptor in body fluids such as blood or cerebrospinal fluid (CSF). This process is known for other proteins such as α-synuclein, Phospho-Tau, β-amyloid, related CNS diseases. At this moment we are currently completing the validation of a system for the detection of the ecto-GPR37 receptor (a N-terminal fragment of the GPR37 produced and liberated after metalloproteinase cleavage) in CSF which could be used as a new biomarker for earlier and finer diagnosis in PD. |
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