Simple SummaryAlzheimer’s disease (AD) is a complex, multifactorial disease where numerous components, such as environment, lifestyle, comorbidities, and genetic predisposition, contribute to triggering the onset of the disease. Several neurobiological brain alterations have been reported during AD pathologies, including the endocannabinoid system (ECS) and associated lipid transmitter-based signaling systems. In this study, we have evaluated the expression levels of the cannabinoid receptors type 2 (CB2) and the novel cannabinoid/lysophospholipid G protein-coupled receptor 55 (GPR55) at different stages of AD. We deeply investigated CB2 and GPR55’s close proximity with Aβ-plaque deposits, as well as neuronal and glial cells, in the ADAppNL-G-Fknock-in mouse model. Additionally, we analyzed whether Aβ42 directly affects CB2 and GPR55 protein expression in neuronal and glial primary cell cultures. Our study shows that the ECS, specifically the CB2 and GPR55 receptors, are altered during AD pathology. Monitoring these receptors may provide new biomarkers for AD diagnosis. CB2 and GPR55 could be potential pharmacological targets for selective compounds to treat AD inflammation.AbstractBackground: The endocannabinoid system (ECS) and associated lipid transmitter-based signaling systems play an important role in modulating brain neuroinflammation. ECS is affected in neurodegenerative disorders, such as Alzheimer’s disease (AD). Here we have evaluated the non-psychotropic endocannabinoid receptor type 2 (CB2) and lysophosphatidylinositol G-protein-coupled receptor 55 (GPR55) localization and expression during Aβ-pathology progression. Methods: Hippocampal gene expression of CB2 and GPR55 was explored by qPCR analysis, and brain distribution was evaluated by immunofluorescence in the wild type (WT) and APP knock-inAppNL-G-FAD mouse model. Furthermore, the effects of Aβ42 on CB2 and GPR55 expression were assessed in primary cell cultures. Results: CB2 and GPR55 mRNA levels were significantly upregulated inAppNL-G-Fmice at 6 and 12 months of age, compared to WT. CB2 was highly expressed in the microglia and astrocytes surrounding the Aβ plaques. Differently, GPR55 staining was mainly detected in neurons and microglia but not in astrocytes. In vitro, Aβ42 treatment enhanced CB2 receptor expression mainly in astrocytes and microglia cells, whereas GPR55 expression was enhanced primarily in neurons. Conclusions: These data show that Aβ pathology progression, particularly Aβ42, plays a crucial role in increasing the expression of CB2 and GPR55 receptors, supporting CB2 and GPR55 implications in AD.