Alterations in Gene and Protein Expression of Cannabinoid CB2 and GPR55 Receptors in the Dorsolateral Prefrontal Cortex of Suicide Victims

Abstract

Recent studies point to the cannabinoid CB₂ receptors (CB₂r) and the non-cannabinoid receptor GPR55 as potential key targets involved in the response to stress, anxiety, and depression. Considering the close relationship between neuropsychiatric disorders and suicide, the purpose of this study was to evaluate the potential alterations of CB₂r and GPR55 in suicide victims. We analyzed gene and protein expression of both receptors by real-time PCR and western blot, respectively, in the dorsolateral prefrontal cortex (DLPFC) of 18 suicide victims with no clinical psychiatric history or treatment with anxiolytics or antidepressants, and 15 corresponding controls. We used in situ proximity ligation assay to evaluate whether the receptors formed heteromeric complexes and to determine the expression level of these heteromers, also assessing the co-expression of heteromers in neurons, astroglia, or microglia cells. CB₂r and GPR55 gene expressions were significantly lower (by 33 and 41%, respectively) in the DLPFC of suicide cases. CB₂r protein expression was higher, as were CB₂-GPR55 heteroreceptor complexes. The results also revealed the presence of CB₂-GPR55 receptor heteromers in both neurons and astrocytes, whereas microglial cells showed no expression. We did not observe any significant alterations of GPR55 protein expression. Additional studies will be necessary to evaluate if these alterations are reproducible in suicide victims diagnosed with different psychiatric disorders. Taken together, the results suggest that CB₂r and GPR55 may play a relevant role in the neurobiology of suicide.

Keywords: CB2r; Dorsolateral prefrontal cortex; GPR55; Gene expression; In situ proximity ligation assay; Suicide.

Fig. 1

CB₂r A isoform and GPR55 gene expression analyses in the DLPFC of suicide victims by real-time PCR. (A. and B.) Total RNA integrity number (RIN) evaluation in the DLPFC of suicide victims and control subjects: (A.) Representative electropherogram images of mean RIN values from DLPFC, (B.) RIN values summary table, gray boxes contain global mean values ± SEM. (C.) Relative gene expression of CB2r A isoform and (D.) GPR55 gene expression in DLPFC of suicide victims (n = 18) and their corresponding control group (n = 15). Asterisks indicate values from suicide victims that are significantly different (p < 0.05) from control subjects. Columns represent the mean and the vertical line ± the standard error of the mean (SEM) of 2^-ΔΔCt

Fig. 2

Protein expression analyses of CB₂r and GPR55 in DLPFC suicide victims by western blot. (A.) Protein expression levels of CB₂r and (B.) GPR55 in DLPFC of suicide victims (n = 18) and their corresponding control group (n = 15). Representative immunoblots of (C.) CB₂r and (D.) GPR55 protein expression. Asterisks indicate values from suicide victims that are significantly different (p < 0.05) from control subjects. Data are expressed as mean ± SEM of arbitrary units (A.U)

Fig. 3

CB₂-GPR55 receptor heteromer expression in human prefrontal cortex. In situ proximity ligation assays were performed as described in Materials and Methods using control (A, A’, A”) and suicidal (C, C’, C”) human brain sections. A” and C” are magnification images of fields (white-dotted rectangles) in, respectively, A’ and C’. Confocal microscopy images (superimposed sections) are shown; heteromers appear as red clusters and Hoechst-stained nuclei appear in blue. (B) Shows the ratio (number of red spots/cell-containing spots) for the indicated samples. Data are the mean ± SEM of counts in 5–7 different fields (each image covered an area of 59,692 μm²) from every sample from controls (n = 7) or suicidal (n = 6). Student’s t test analysis showed significant inter-group differences on ratio of PLA spots. Asterisks indicate values from suicide victims that are significantly different (p < 0.05) from control subjects. (D) A negative control obtained by omitting one of the primary antibodies is shown. Scale bars: 50 μm (A, A’, C, C′), 10 μM (A”, C”)

Fig. 4

In situ proximity ligation-based detection of cellular types expressing CB₂-GPR55 receptor heteromers. Panel 1A–1D: Expression of CB₂-GPR55 receptor heteromers (PLA-detected, red channel) in astrocytes (GFAP immunostain; green channel). TOPRO3-counterstain was used to disclose cell nuclei. 1B and 1D are insets taken from 1A and 1C, respectively, to illustrate the expression of CB₂r-GPR55 receptor heteromers in astrocytes. Cortical neurons also expressed heteromeric complexes made of CB₂r and GPR55 receptors. Panels 1A–1B were taken from control samples whereas panels 1C–1D belong to suicide cases. Panel 2A–2D: Expression of CB₂r-GPR55 receptor heteromers (PLA-detected, red channel) in microglial cells (Iba1 immunostain; green channel). TOPRO3-counterstain was used to disclose cell nuclei. 2B and 2D are insets taken from 2A and 2B, respectively, to properly illustrate the expression of CB₂r-GPR55 receptor heteromers in microglial cells. Cortical neurons also expressed heteromeric complexes made of CB₂r and GPR55 receptors. Panels 1A–1B were taken from control samples whereas panels 2C–D belong to suicide cases. Scale bars are 800 μm for low-magnification panels and 10 μm for insets. Panel 3A–3D: Identification of CB₂r-GPR55 receptor heteromers in layer V cells of the PFC cortex of control samples (3A–3C) and suicide patients (3D–3F). CB₂r-GPR55 heteromers were observed both in neurons (labeled as N) as well as in glial cells (G; presumed astrocytes). Scale bar is 10 μm in all panels