Relevant Molecular and Functional G.Protein Coupled Receptor Interactions in Neuroinflammation and Addiction = Interaccions moleculars i funcionals de receptors acoblats a proteïna G rellevants en neuroinflamació i addició

Abstract

G-protein coupled receptors (GPCR) constitute a complex and versatile superfamily of membrane receptors with relevance in maintaining homeostasis. The thesis project has focused in members of this superfamily: the CB1 and CB2 cannabinoid receptors, which are part of the endocannabinoid system, and the ghrelin receptor GHS- R1a.

The endocannabinoid system regulates, in mammals, body temperature, neurotransmission or immune response among others. Indeed, the endocannabinoid system has been related to processes coursing with neuroinflammation, in particular those that are derived from neurodegenerative diseases like Alzheimer’s disease (AD) or Parkinson’s disease (PD). In this thesis, it has been investigated the role of CB1-CB2 receptor heteromers in a neuroinflammatory context. Both in an immortalised microglial cell line, N9, and microglial primary cultures, there is an increase of CB2R and CB1-CB2 heteromer expression upon treatment of the cells with LPS and INF-. Interestingly, the characteristic negative cross-talk of CB1-CB2 receptor heteromers becomes positive after the treatment. Such change in the cannabinoid receptor signalling was observed in an animal model of the AD. In addition, in the hemilesioned rat model of PD, the administration of levodopa and acquisition of dyskinesia leads to a significant increase in CB1-CB2 heteroreceptor expression.

GPCR activity can be regulated by interaction with diverse proteins. The thesis project has focused in the potential interaction of the cannabinoid CB1R with calcium- binding proteins. These calcium sensor proteins modulate cell events depending on the concentration of cytosolic Ca2+. First of all, an interaction of NCS1 and calneuron1 (but not caldendrin) calcium sensors with the receptor has been established. At low intracellular calcium levels, the predominant interaction occurs between CB1R and NCS1, while at high intracellular calcium levels CB1R-calneuron1 interactions are more likely to occur. At the functional level, the cAMP signalling of CB1R is abolished when calneuron1 is interacting with the receptor, whereas the CB1R-NCS1 interaction does not promote any signalling disturbance in the cAMP-mediated pathway.

Whereas GHS-R1a is a GPCR, an alternative splicing of the gene that codes for the receptor generates the GHS-R1b isoform. This is a truncated protein that lacks the 6th and 7th transmembrane domains: it is unable to bind ligands or to signal. In this thesis, it has been addressed the role of the GHS-R1b isoform. The results show that the GHS-R1b isoform enables GHS-R1a expression at the plasma membrane due to heteromerization. Moreover, GHS-R1b can allosterically modulate GHS-R1a. When there is a low GHS- R1b/GHS-R1a expression ratio, GHS-R1a is functional, but at high GHS-R1b/GHS-R1a expression ratio, GHS-R1b negatively modulates GHS-R1a activity. These results obtained in a heterologous expression system were confirmed in striatal and hippocampal neuron cultures.

GHS-R1a, a receptor expressed in the reward pathway, exerts a role in controlling food intake. In this thesis project, a molecular relationship between GHS-R1a and cocaine consumption has been discovered. First of all, an interaction of GHS-R1a and sigma-1 receptor was demonstrated. Secondly, cocaine binding to sigma-1 receptor leads to a

negative modulation of GHS-R1a signalling. Moreover, GHS-R1a-sigma-1 interactions are increased in rats after acute cocaine consumption, while its presence in rats chronically treated with cocaine is similar to that in controls. A molecular mechanism is suggested through which cocaine, via GHS-R1a/sigma-1 receptor complexes, can influence food consumption.

Els receptors CB1 i CB2 de cannabinoides i el GHS-R1a de grelina pertanyen a la superfamilia de receptors acoblats a proteïnes G (GPCR).

Els endocannabinoids i els receptors CB1 i CB2 tenen una implicació en la regulació de la neuroinflamació. L’activació de la micròglia altera la senyalització cannabinoide i incrementa l’expressió del receptor CB2 i l’heteròmer CB1-CB2. L’heteròmer és present en micròglia d’un model de la malaltia de Parkinson i en micròglia d’un model de la malaltia d’Alzheimer.

Els sensors de calci NCS1 i calneuron1 són capaços d’interaccionar amb el receptor CB1. Mentre que a elevades concentracions intracel·lulars de calci citosòlic, predomina la interacció CB1R-Calneuon1 i es provoca la inhibició de la senyalització per AMPc del receptor, a baixes concentracions de calci, predomina la interacció CB1R-NCS1, i no hi ha conseqüències a nivell de senyalització via AMPc.

La expressió del gen del receptor de grelina dóna lloc al receptor GHS-R1a i a una forma truncada (GHS-R1b) en la que manquen els dominis TM6 i TM7. En aquesta tesi es descriu el paper regulador de GHS-R1b sobre GHS-R1a, promovent, mitjançant heteromerització, el tràfic cap a membrana de GHS-R1a i generant una regulació al·lostèrica negativa.

Finalment, s’ha observat que GHS-R1a interacciona amb el receptor sigma-1, provocant conseqüències funcionals. A més, la unió de la cocaïna al receptor sigma-1 redueix l’efecte de la grelina mitjançada pel seu receptor. Aquesta interacció proporciona un mecanisme molecular que explicaria per què la cocaïna disminueix la sensació de gana. La interacció es dóna en rates tractades de forma aguda amb cocaïna, suggerint una relació molecular entre el consum de cocaïna i la regulació de la ingesta.