Antagonization of OX1 Receptor Potentiates CB2 Receptor Function in Microglia from APP(Sw/Ind) Mice Model

Microdialysis assays demonstrated a possible role of orexin in the regulation of amyloid beta peptide (A beta) 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 (R) SureFire (R) 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.

Automated summary

The study demonstrates the interaction between orexin and CB2R in Alzheimer's disease animal models, forming the CB2-OX1 receptor complex, which may serve as a new therapeutic target for Alzheimer's disease.