Fig 2: Generation and molecular characterization of Gpr55+/- mice.(A) Schematic of the CRISPR/Cas9 strategy to delete Gpr55. The binding sites of the CRISPR/Cas9 guide RNAs are noted as sgRNA1 and sgRNA2. The arrows show binding sites of the genotyping primers. Schematic created using BioRender.com. (B) DNA genotyping gel showing wildtype (WT), Gpr55+/- (+/-) and Gpr55-/- progeny (-/-) with a no DNA control (Blank). (C) Relative Gpr55 transcript levels in the hippocampus of wildtype, Gpr55+/- and Gpr55-/- mice, revealing a complete loss of Gpr55 expression in homozygous knockouts. Gpr55 expression was measured in primary RNA pools using RT-ddPCR and expressed as a ratio of Tbp. Data represents mean ± SEM, with n = 5–8 per group (WT: 3 males, 2 females; Gpr55+/-: 4 males, 4 females; Gpr55-/-: 2 males, 3 females).

Fig 3: Astrocytes and microglia GPR55 immunoreactivity in 12-month-old AppNL-G-F mice. (a) Representative immunostaining reveals a lack of overlapping staining between astrocytes marker (GFAP, red) and GPR55 (green) in a 12-month-old AppNL-G-F mouse. (b) Representative images showing overlapping staining observed between microglia marker (Iba1, red) and GPR55 receptor (green). (c) Marked-activated microglia revealed by Iba1 (red) were found around amyloid plaques (FSB, blue). Abbreviations: GFAP: Glial fibrillary acidic protein, FSB: 1-Fluoro-2,5-bis(3-carboxy-4-hydroxystyryl) benzene, DAPI: (4′,6-diamidino-2-phenylindole).

Fig 4: Expression of ECS components in female mouse reproductive tract during aging. Protein expression levels of ECS receptors (CB1, CB2, GPR55, TRPV1), AEA metabolic enzymes (NAPE-PLD, FAAH), and 2-AG metabolic enzymes (DAGL-α, DAGL-β, MAGL) detected in ovaries (a), oviducts (b), and uteri (c) collected from prepubertal (PrP), adult (Ad), late reproductive (LR), and post-reproductive (PR) mice. Results of three independent experiments expressed as mean OD values ± standard error of the mean (SEM). Letters indicate statistical significance (p < 0.05) as follows: a: PrP vs. Ad, b: PrP vs. LR; c: PrP vs. PR; d: Ad vs. LR; e: Ad vs. PR; f: LR vs. PR. For more detailed information, please refer to Table S1 in the Supplementary Materials.

Fig 5: Strain-specific Gpr55 expression.Relative Gpr55 transcript levels in the (A) cortex and (B) hippocampus of wildtype (WT, blue bars) and Scn1a+/- (gray bars) mice on 129 and F1 (129 x B6) background strains. Gpr55 expression was measured in primary RNA pools using RT-ddPCR and are expressed as a ratio of Tbp. Each primary pool includes tissue from 3–4 mice, with at least one from each sex. Data represents mean ± SEM, with n = 7–11 pools per group. Cortical and hippocampal samples were dissected and pooled from different cohorts of F1.Scn1a+/- mice. Cortical and hippocampal Gpr55 expression was significantly greater in F1 compared to 129 mice (****p < 0.0001, two-way ANOVA). Scn1a+/- mice expressed significantly greater Gpr55 than wildtype mice on the F1 (129 x B6) background strain (*p < 0.05 two-way ANOVA followed by Sidak’s test).