Antagonization of OX1 Receptor Potentiates CB2 Receptor Function in Microglia from APPSw/Ind Mice Model

Iu Raïch; Joan Biel Rebassa; Jaume Lillo; Arnau Cordomi; Rafael Rivas-Santisteban; Alejandro Lillo; Irene Reyes-Resina; Rafael Franco; Gemma Navarro

doi:10.3390/ijms232112801

Antagonization of OX₁ Receptor Potentiates CB₂ Receptor Function in Microglia from APP_Sw/Ind Mice Model

Int J Mol Sci. 2022 Oct 24;23(21):12801. doi: 10.3390/ijms232112801.

Authors

Iu Raïch^{1

2

3}, Joan Biel Rebassa^{1

2

3}, Jaume Lillo^{2

3}, Arnau Cordomi⁴, Rafael Rivas-Santisteban^{2

3}, Alejandro Lillo^{1

2

3}, Irene Reyes-Resina^{1

2

3}, Rafael Franco^{1

5}, Gemma Navarro^{1

2

3}

Affiliations

¹ Molecular Neuropharmacology Laboratory, Department of Biochemistry and Physiology, School of Pharmacy and Food Science, Universitat de Barcelona, 08007 Barcelona, Spain.
² CiberNed, Network Center for Neurodegenerative Diseases, National Spanish Health Institute Carlos III, 28029 Madrid, Spain.
³ Neurosciences Institut, University of Barcelona (NeuroUB), 08028 Barcelona, Spain.
⁴ Bioinformatics, ESCI-UPF, 08003 Barcelona, Spain.
⁵ School of Chemistry, Universitat de Barcelona, 08007 Barcelona, Spain.

Abstract

Microdialysis assays demonstrated a possible role of orexin in the regulation of amyloid beta peptide (Aß) levels in the hippocampal interstitial fluid in the APP transgenic model. CB₂R is overexpressed in activated microglia, showing a neuroprotective effect. These two receptors may interact, forming CB₂-OX₁-Hets and becoming a new target to combat Alzheimer's disease. Aims: Demonstrate the potential role of CB₂-OX₁-Hets expression and function in microglia from animal models of Alzheimer's disease. Receptor heteromer expression was detected by immunocytochemistry, bioluminescence resonance energy transfer (BRET) and proximity ligation assay (PLA) in transfected HEK-293T cells and microglia primary cultures. Quantitation of signal transduction events in a heterologous system and in microglia cells was performed using the AlphaScreen^® SureFire^® kit, western blot, the GCaMP6 calcium sensor and the Lance Ultra cAMP kit (PerkinElmer). The formation of CB₂-OX₁ receptor complexes in transfected HEK-293T cells has been demonstrated. The tetrameric complex is constituted by one CB₂R homodimer, one OX₁R homodimer and two G proteins, a G_i and a G_q. The use of TAT interfering peptides showed that the CB₂-OX₁ receptor complex interface is TM4-TM5. At the functional level it has been observed that the OX₁R antagonist, SB334867, potentiates the action induced by CB₂R agonist JWH133. This effect is observed in transfected HEK-293T cells and microglia, and it is stronger in the Alzheimer's disease (AD) animal model APPSw/Ind where the expression of the complex assessed by the proximity ligation assay indicates an increase in the number of complexes compared to resting microglia. The CB₂-OX₁ receptor complex is overexpressed in microglia from AD animal models where OX₁R antagonists potentiate the neuroprotective actions of CB₂R activation. Taken together, these results point to OX₁R antagonists as drugs with therapeutic potential to combat AD. Data access statement: Raw data will be provided by the corresponding author upon reasonable requirement.

Keywords: Alzheimer’s disease; activated microglia; cannabinoids; orexin.

MeSH terms

Alzheimer Disease* / metabolism
Amyloid beta-Peptides / metabolism
Animals
Disease Models, Animal
Mice
Mice, Transgenic
Microglia* / metabolism
Receptor, Cannabinoid, CB2 / genetics
Receptor, Cannabinoid, CB2 / metabolism

Substances

Amyloid beta-Peptides
Receptor, Cannabinoid, CB2

Grants and funding

PID2020-113430RB-I00/MINISTERIO DE CIENCIA E INNOVACIÓN