Thèse Rôle de la Composition Lipidique Membranaire dans le Contrôle du Trafic et de la Signalisation des Récepteurs de la Dopamine dans les Neurones de l'Analyse Cellulaire aux Conséquences sur le H/F - 33

Établissement : Université de Bordeaux
École doctorale : Sciences de la Vie et de la Santé
Laboratoire de recherche : Institut Interdisciplinaire de Neurosciences
Direction de la thèse : David PERRAIS ORCID 0000000258785408
Début de la thèse : 2026-10-01
Date limite de candidature : 2026-05-20T23:59:59

Le degré de poly-insaturation des phospholipides membranaires influence de manière cruciale les propriétés biophysiques des membranes et des protéines membranaires. Le cerveau est fortement enrichi en acides gras polyinsaturés (AGPI) et leur carence a été associée à plusieurs troubles neuropsychiatriques. Il est important de noter que le récepteur de la dopamine D2 (D2R), un récepteur couplé aux protéines G (RCPG), est souvent touché dans ces troubles et représente la cible principale des antipsychotiques. Nous avons précédemment montré que le dysfonctionnement du système de récompense chez les animaux déficients en AGPI n-3 est associé à une activité D2R altérée. En outre, au niveau cellulaire, nous avons montré dans les cellules HEK293, une lignée cellulaire humaine largement utilisée pour étudier la signalisation et le trafic des RCPG, que l'enrichissement en AGPI affecte fortement et spécifiquement l'internalisation du D2R induite par le ligand. Nous avons identifié des résidus clés dans la face cytosolique du D2R qui sont responsables de cette sensibilité. L'étape suivante consiste à déterminer l'effet des niveaux d'AGPI sur l'internalisation et la signalisation du D2R dans les neurones. Pour ce faire, nous utiliserons une combinaison de techniques d'imagerie avancées pour suivre le trafic et la signalisation des récepteurs à l'aide de sondes fluorescentes. Nous définirons et validerons des protocoles pour contrôler les niveaux d'AGPI dans les neurones en culture et déterminerons leur effet sur la signalisation par les récepteurs de la dopamine insensibles et sensibles aux AGPI à une résolution subcellulaire. Enfin, nous exprimerons des récepteurs de type sauvage et mutant in vivo pour évaluer leurs effets sur les propriétés neuronales dans des conditions physiologiques. Enfin, nous évaluerons les conséquences sur le traitement de la récompense.

The degree of poly-unsaturation in membrane phospholipids critically influences the biophysical properties of both membranes and membrane proteins. The brain is highly enriched in poly-unsaturated fatty acids (PUFAs) and their deficiency has been associated with several neuropsychiatric disorders. Importantly, the Dopamine D2 receptor (D2R), a class A G protein coupled receptor (GPCR), is consistently impacted in these disorders and represents the main target of antipsychotics1. The co-director Pierre Trifilieff (PT) has shown that deficits in motivation of n-3 PUFA deprived mice is related to a dysfunction of D2R-expressing striatal projection neurons2, and that n-3 PUFA deficiency decreases the sensitivity to D2R antagonists in vivo3. These results reveal a strong interplay between the levels of PUFAs in brain cells and D2R signaling.
To analyze this link, the two co-directors, PT and David Perrais (DP), in collaboration with the team of Isabel Alves (CBMN, Pessac), have set up a protocol of PUFA enrichment in D2R expressing HEK293 cells (a human cell line widely used to study GPCR signalling and trafficking). They found that binding of ligands and signalling are affected in cells enriched in the n-3 PUFA docosahexaenoic acid (DHA) or the n-6 PUFA docosapentaenoic acid (DPA)3. Moreover, after ligand binding, most GPCRs, including D2R, are quickly and strongly internalized into endosomes which modulates the extent and nature of the signalling events they generate4. They showed that the endocytosis of D2R following ligand binding is strongly modulated by both PUFAs DHA and DPA in HEK293 cells5. This effect is specific for D2R endocytosis, as it does not affect the endocytosis of 7 other class A GPCRs including the D1R. Finally, mutating two consecutive serine residues in the cytosolic face of D2R into alanines abolishes D2R sensitivity to PUFAs without affecting its ability to undergo endocytosis.
We now need to determine to what extent PUFAs in neuronal cells affect dopaminergic signaling and trafficking. D2R was shown to internalize following activation by agonists in neurons in culture6 and in acute brain slices7,8. Interestingly, the degree of internalization varies between neuronal types8 and PUFA levels are different in various brain tissues9. These differences suggest that D2R endocytosis following activation could be differentially affected by PUFA composition between neuronal subtypes. Although brain PUFA levels and composition can be regulated in vivo, mainly by the diet, PUFA levels can easily be controlled in cultures of primary neurons10,11.
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Our primary objective is to test the hypothesis that the endocytosis and signaling of D2R, and perhaps D3R (also predicted to be sensitive to PUFAs), can be specifically controlled by PUFA levels in neurons, while other receptors such as D1R are presumably insensitive. Moreover, we will use the PUFA insensitive mutant D2R(S147,148A) to test for alterations in trafficking and signaling in vivo, and its consequences on reward processing in mice.

We will first establish a protocol to assess and control PUFA levels in cultured neurons from various brain areas (hippocampus, cortex, striatum). It is currently unclear what are the fractions of fatty acids composing phospholipids in cultured neurons10,11. This likely depends on the conditions of culture, and can be modulated by incubation with various fatty acids, such as docosahexaenoic acid (DHA), the main n-3 PUFA in the brain.
We will analyze PUFA composition of phospholipids in cultured neurons with the Plateforme Metabolome of Bordeaux (https://metabolome.u-bordeaux.fr/) in conditions of PUFA enrichment or not.
We will measure the internalization of labelled D1, D2 and D3 dopamine receptors in the dendrites, soma and axon of neurons with fixed cell and live cell fluorescence imaging, such as the ppH protocol, established by the director DP, which enables the detection of single endocytic vesicles5,12,13. We will measure the internalization of wild-type and mutated D2 receptor in conditions of PUFA enrichment.
We will measure the signaling events induced by dopamine receptor activation (recruitment of b-arrestin, increase in cyclic AMP, activation of protein kinase A)14,15 in conditions of PUFA enrichment.
We will determine the effect of expression of wild-type or mutated D2 receptor in the striatum on neuronal properties and sensitivity to dopamine through ex vivo electrophysiological analyses. Ultimately, the consequence on reward processing will be assessed through behavioral analyses based on operant conditioning paradigms.