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. 2024 Apr 10;25(8):4176.
doi: 10.3390/ijms25084176.

Heteromers Formed by GPR55 and Either Cannabinoid CB1 or CB2 Receptors Are Upregulated in the Prefrontal Cortex of Multiple Sclerosis Patients

Carlota Menéndez-Pérez  1   2   3 , Rafael Rivas-Santisteban  4   5   6 , Eva Del Valle  1   2   3 , Jorge Tolivia  1   2   3 , Ana Navarro  1   2   3 , Rafael Franco  4   5 , Eva Martínez-Pinilla  1   2   3
Affiliations

Heteromers Formed by GPR55 and Either Cannabinoid CB1 or CB2 Receptors Are Upregulated in the Prefrontal Cortex of Multiple Sclerosis Patients

Carlota Menéndez-Pérez et al. Int J Mol Sci. .

Abstract

Multiple sclerosis (MS) is an autoimmune, inflammatory, and neurodegenerative disease of the central nervous system for which there is no cure, making it necessary to search for new treatments. The endocannabinoid system (ECS) plays a very important neuromodulatory role in the CNS. In recent years, the formation of heteromers containing cannabinoid receptors and their up/downregulation in some neurodegenerative diseases have been demonstrated. Despite the beneficial effects shown by some phytocannabinoids in MS, the role of the ECS in its pathophysiology is unknown. The main objective of this work was to identify heteromers of cell surface proteins receptive to cannabinoids, namely GPR55, CB1 and CB2 receptors, in brain samples from control subjects and MS patients, as well as determining their cellular localization, using In Situ Proximity Ligation Assays and immunohistochemical techniques. For the first time, CB1R-GPR55 and CB2R-GPR55 heteromers are identified in the prefrontal cortex of the human brain, more in the grey than in the white matter. Remarkably, the number of CB1R-GPR55 and CB2R-GPR55 complexes was found to be increased in MS patient samples. The results obtained open a promising avenue of research on the use of these receptor complexes as potential therapeutic _targets for the disease.

Keywords: PLA; cannabinoids; endocannabinoid system; oligomerization; prefrontal cortex.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
CB1R-GPR55 heteromers detected using the In Situ Proximity Ligation Assay (PLA) in the grey (GM) and white matter (WM) of the prefrontal cortex of samples from the controls and multiple sclerosis (MS) patients. Representative confocal images (40×) showing PLA label for CB1R-GPR55 heteromers as red dots in cells with DAPI-stained nuclei. Scale bar 50 µm (A). Quantification of CB1R-GPR55 heteromers as the number of red dots per cell in the GM and WM in the prefrontal cortex samples of the controls and MS patients. Data are the mean ± SEM (40 fields per section) (B). Significant differences were analyzed via two-way ANOVA followed by post hoc Bonferroni’s test. **** p < 0.0001 compared with GM; # p < 0.05, #### p < 0.0001 compared with the control.
Figure 2
Figure 2
CB2R-GPR55 heteromers detected using the In Situ Proximity Ligation Assay (PLA) in the grey (GM) and white matter (WM) of the prefrontal cortex of samples from the controls and multiple sclerosis (MS) patients. Representative confocal images (40×) showing the PLA label for CB2R-GPR55 heteromers as red dots in cells with DAPI-stained nuclei. Scale bar 50 µm (A). Quantification of CB2R-GPR55 heteromers as the number of red dots per cell in the GM and WM in the prefrontal cortex samples of the controls and MS patients. Data are the mean ± SEM (40 fields per section) (B). Significant differences were analyzed via two-way ANOVA followed by post hoc Bonferroni’s test. **** p < 0.0001 compared with GM; # p < 0.05, #### p < 0.0001 compared with the control.
Figure 3
Figure 3
Expression of CB1R-GPR55 heteromers in neurons, astrocytes, oligodendrocytes, and microglia in the prefrontal cortex of the control subjects. Following the chromogenic immunohistochemical detection of the four different types of cells, those positive for NF-200, GFAP, αβ-crystallin, and iba-1 (A,D,G,J), CB1R-GPR55 heteromers were identified as red dots in cells with DAPI-stained nuclei using the PLA method (B,E,H,K). Digital overlays of PLA images (red fluorescent signal) and DAB immunolabeling images (the DAB signal converted into a green color) clearly show that only neurons express CB1R-GPR55 receptor complexes (arrows), whereas oligodendrocytes, astrocytes, and microglia completely lack them (C,F,I,L). 40×. Detail: 100×. Scale bar 50 µm.
Figure 4
Figure 4
Expression of CB2R-GPR55 heteromers in neurons, oligodendrocytes, astrocytes, and microglia in the prefrontal cortex of the control subjects. Following the chromogenic immunohistochemical detection of the four different types of cells, those positive for NF-200, GFAP, αβ-crystallin, and iba-1 (A,D,G,J), CB2R-GPR55 heteromers were identified as red dots in cells with DAPI-stained nuclei using the PLA method (B,E,H,K). Digital overlays of PLA images (red fluorescent signal) and DAB immunolabeling images (the DAB signal converted into a green fluorescent signal) clearly showed that only neurons express CB2R-GPR55 receptor complexes (arrows), whereas oligodendrocytes, astrocytes, and microglia completely lack them (C,F,I,L). 40×. Detail: 100×. Scale bar 50 µm.
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