Evidence of decreased gap junction coupling between astrocytes and oligodendrocytes in the anterior cingulate cortex of depressed suicides

Abstract

Glial dysfunction is a major pathophysiological feature of mood disorders. While altered astrocyte (AS) and oligodendrocyte-lineage (OL) functions have been associated with depression, the crosstalk between these glial cell types has never been assessed in that context. AS are potent regulators of myelination, in part through gap junction (GJ) channels formed by the heterotypic coupling of AS-specific (Cx30 and Cx43) and OL-specific (Cx32 and Cx47) connexins. This study therefore aimed at addressing the integrity of AS/OL coupling in the anterior cingulate cortex (ACC) of depressed suicides. Using immunofluorescence and confocal imaging, we characterized the distribution of Cx30 and mapped its expression onto OL somas, myelinated axons, and brain vasculature in postmortem brain samples from depressed suicides (N = 48) and matched controls (N = 23). Differential gene expression of key components of the GJ nexus was also screened through RNA-sequencing previously generated by our group, and validated by quantitative real-time PCR. We show that Cx30 expression localized onto OL cells and myelinated fibers is decreased in deep cortical layers of the ACC in male-depressed suicides. This effect was associated with decreased expression of OL-specific connexins, as well as the downregulation of major connexin-interacting proteins essential for the scaffolding, trafficking, and function of GJs. These results provide a first evidence of impaired AS/OL GJ-mediated communication in the ACC of individuals with mood disorders. These changes in glial coupling are likely to have significant impact on brain function, and may contribute to the altered OL function previously reported in this brain region.

Fig. 1

Distribution of connexin 30 immunoreactivity in the human anterior cingulate cortex. a Cx30 (red) at low (top panels) and high (bottom panels) magnification shows a strong level of expression in cortical gray matter but virtually no expression in deep white matter, with characteristic puncta-like immunoreactivity enriched in the neuropil. Myelin Proteolipid Protein (PLP) (green) was used to delineate white matter and visualize myelinated fibers. The area between dashed lines corresponds to deep cortical layers (layers 5–6) in which further quantification was performed. Cx30 surrounds and colocalizes with myelinated axons in cortical gray matter. Scale bars = 200 µm (top) and 2 µm (bottom). b Connexin 30 (red) is heavily enriched along brain vasculature and perivascular endfeet of astrocytes stained with vimentin (green). Maximum intensity Z projection at high magnification (bottom panels) shows strong colocalization of Cx30 puncta along blood vessels and perivascular endfeet, consistent with previous description of Cx30 distribution in the brain. Scale bars = 100 µm (top) and 10 µm (bottom). c Top panel shows the enrichment of Cx30 puncta labeling (red) along Glial fibrillary acidic protein (GFAP+) astrocytic endfeet (green) lining a blood vessel (*). Bottom panel shows another blood vessel surrounded by GFAP+ astrocytic fibers and perivascular endfeet (green), with strong colocalization of Cx30 and Cx43 (blue), as expected from the literature. Scale bars = 5 µm

Fig. 2

Depressed Suicides show no change in the density of Cx30 puncta compared with controls in the anterior cingulate cortex. a Density of total Cx30 puncta in the ACC of depressed suicides without (DS-nCA, N = 5) or with (DS-CA, N = 7) a history of child abuse and Controls (N = 11). No difference between groups was found (Kruskal–Wallis ANOVA: H(2,23) = 0.3268, P = 0.84). b No effect of substance abuse was found on the density of Cx30 in depressed suicides. Depressed suicides without (DS-nSub, N = 7) or with (DS-Sub, N = 5) a history of substance abuse showed similar densities of Cx30 compared with controls (N = 11) (Kruskal–Wallis ANOVA: H(2,23) = 0.02247, P = 0.99). c Previous medication with antidepressants had no effect on the densities of Cx30 in the ACC. Depressed suicides without (DS-nMed, N = 8) or with (DS-Med, N = 4) a known antidepressant prescription the last 3 months before death showed similar densities of Cx30 compared with controls (N = 11) (Kruskal–Wallis ANOVA: H(2,23) = 0.3308, P = 0.85). d No correlation between Cx30 puncta densities and postmortem interval (P = 0.63) or e age (P = 0.33) was found, indicating overall that the absence of changes in Cx30 puncta density between cases and controls is likely not linked to covariables. Data represent mean ± s.e.m

Fig. 3

Specific decrease in Cx30 puncta localized onto oligodendrocytes and myelinated fibers in the anterior cingulate of depressed suicides. a Drawing illustrating astrocyte-oligodendrocyte (AS/OL) GJ coupling at the soma and around myelinated axons through heterotypic pairing of Cx30 or Cx43 on the astrocyte (colored in green) side, with Cx32 and Cx47, respectively on the oligodendrocyte (colored in red) side. Adapted from Orthmann-Murphy et al. [47]. b Changes in AS/OL pairing between controls and depressed suicides was first addressed through quantification of Cx30 puncta (green) localized onto oligodendrocytes cells bodies stained with Nogo-A (red). Some Cx30 immunoreactivity is found surrounding oligodendrocytes cells bodies and colocalizing in this example with GFAP+ astrocytes (gray). Arrowheads point to Cx30 puncta specifically localized onto Nogo-A+ cells (see Fig. S4). Scale bar = 1 µm. c Depressed suicides, regardless of history of child abuse, showed a decrease in Cx30 puncta localized onto Nogo-A+ oligodendrocytes (Kruskal–Wallis ANOVA: H(2,23) = 9.09, P < 0.05; Dunn’s multiple comparisons tests: Controls (N = 11) vs DS-nCA (N = 5) and Controls vs DS-CA (N = 7), P < 0.05). d Because history of CA had no effect on Cx30 puncta localized onto NogoA+ cells, we pooled both depressed suicides groups to address the potential contribution of confounding factors on changes observed between Controls (N = 11) and Depressed Suicides (N = 12). After controlling for age, PMI, pH, substance dependence, and antidepressant medication, group (MDD/suicide) was still found to be the only significant factor (Table S3), with Depressed Suicides showing decreased Cx30 puncta localized onto Nogo-A+ cells (F(1,23) = 7675, P = 0.014). e Changes in AS/OL pairing between controls and depressed suicides was also assessed though quantification of Cx30 puncta (green) localized onto myelinated axons visualized with Myelin Proteolipid Protein (PLP, red). Arrowheads indicate Cx30 puncta directly considered as localized onto PLP fibers, based on the approach described in Fig. S5. Scale bar = 0.5 µm. f We found differences between groups (Kruskal–Wallis ANOVA: H(2,27) = 7.471, P < 0.05), with Cx30 puncta densities decreased along PLP+ myelinated fibers in depressed suicides regardless of history of CA, although differences were more statistically robust in DS-CA (Dunn’s multiple comparison test, Controls (N = 10) vs DS-CA (N = 11): P < 0.05), as only a trend was detectable between DS-nCA and controls (Dunn’s multiple comparison test, Controls vs DS-nCA (N = 6): P < 0.1). Densities of Cx30 puncta localized onto PLP+ myelinated axons were however similar between DS-CA and DS-nCA (Dunn’s multiple comparison test, DS-nCA vs DS-CA: P = 0.99). g Because history of CA had no effect on Cx30 puncta density localized onto PLP+ fibers, we pooled both depressed suicides groups to address the potential contribution of confounding factors on changes observed between Controls (N = 10) and Depressed Suicides (N = 17). After controlling for age, PMI, pH, substance dependence, and antidepressant medication, group (MDD/suicide) was still found to be the only significant factor (Table S4), with Depressed Suicides showing decreased Cx30 puncta density localized onto PLP+ fibers (F(1,27) = 5.755, P = 0.026). h To address the specificity of the observed changes in Cx30 puncta localized onto OL cells or myelinated fibers, we measured Cx30 puncta (green) density localized onto blood vessels stained with laminin (red). Arrowheads point to Cx30 puncta directly considered as localized onto blood vessels, based on the approach described in Fig. S6. Scale bar = 2 µm. i Changes in Cx30 puncta localization seem specific to AS/OL GJ coupling since no change between groups (Controls, N = 8; DS-nCA, N = 8; DS-CA, N = 9) was observed in Cx30 puncta (green) localized onto blood vessels stained with laminin (red) (Kruskal–Wallis ANOVA: H(2,25) = 0.2, P = 0.9). j After controlling for age, PMI, pH, substance dependence, and antidepressant medication, group (MDD/suicide) was still not found to be affecting Cx30 puncta density localized onto blood vessels (Table S5, F(1,25) = 0.003, P = 0.960). Scale bar = 2 µm. *P < 0.05; **P < 01; #, 0.05 < P < 0.1 (versus Controls). Data in represent mean ± s.e.m

Fig. 4

Genes involved in the scaffolding, trafficking, and function of gap junction channels and cell–cell junctions are differentially expressed in the anterior cingulate cortex of depressed suicides. a RNA-sequencing results comparing samples from the anterior cingulate cortex of depressed suicides with a history of child abuse vs controls were screened to probe the differential expression of a literature-based list of genes related to GJ channel scaffolding and function. We found that among genes coding for major connexins, depressed suicides showed a downregulation specifically for the two oligodendrocyte-specific connexins Cx32 (GJB1) and Cx47 (GJC2). Major tight junction proteins (TJPs) such as occluding (OCLN), zona occludens-1 and -2 (TJP1/ZO-1 and TJP2/ZO-2), as well as claudin-11/OSP (CLDN11), an essential component of myelin interlamellar tight junctions were also found to be downregulated in depressed suicides. Genes coding for connexins-binding partners and regulators of GJ-mediated intercellular communication, such as caveolin-1 and -2 (CAV1 and CAV2) and alpha/beta catenins (CTNNA1 and CTNNB1) were also found to be downregulated. Of note, several genes coding for actin-binding proteins thought to bridge connexins to actin microfilament cytoskeleton, such as drebrin (developmentally regulated brain protein, DBN1) and spectrins were found to be collectively upregulated in depressed suicides compared with controls. Red and green bars indicate significant downregulation and upregulation, respectively, while gray bars indicate no significant change in gene expression. The drawing on the right depicts the suggested interactions between these different components of the GJ nexus and connexin-interacting proteins, adapted from Dbouk et al. [70]. Red and green indicate components found to be downregulated or upregulated, respectively, in the RNA-sequencing dataset. b Because the RNA-sequencing dataset was generated comparing samples from controls and depressed suicides with a history of child abuse (DS-CA), a subset of these differentially expressed genes was selected for validation with quantitative RT-PCR and adding a third group of depressed suicides with no history of child abuse (DS-nCA). Globally and consistent with the RNA-sequencing results, DS-CA subjects showed a downregulation of Cx32/GJB1, Cx47/GJC2, CAV1 and CAV2, OCLN, and an upregulation of DBN1 (Controls (N = 16–23) vs DS-CA (N = 19–25), *P < 0.05, **P < 0.01, ^#P < 0.1), although these results were not as statistically robust as observed in the RNA-sequencing dataset. Importantly these changes did not seem specific to a history of child abuse, as no significant difference was found between DS-CA and DS-nCA for any of these genes, while DS-nCA showed similar trends towards decreased expression of Cx32/GJB1, Cx47/GJC2, CAV2, and OCLN (Controls (N = 16–23) vs DS-nCA (N = 12–24), ^#P < 0.1). c Since changes in gene expression were not found to be specific to a history of child abuse, we combined both depressed suicides groups to increase our statistical power and perform backward stepwise linear regressions and tease out the contributions of confounding variables in differential gene expression (Tables S6–S13). Depressed Suicides showed significant downregulations of oligodendrocyte connexins Cx32/GJB1 and Cx47/GJC2, CAV1 and CAV2 as well as OCLN (Controls (N = 16–23) vs Depressed Suicides (N = 33–48), *P < 0.05, **P < 0.01). The upregulation of DBN1 observed in the RNA-seq dataset was however not validated, although a small trend towards increased expression was detectable (Controls (N = 23) vs Depressed Suicides (N = 36), P = 0.13). Data represent mean ± s.e.m. TJPs tight junction proteins, GJB6 gap junction protein beta 6 (Cx30), GJA1 gap junction protein alpha 1 (Cx43), GJB1 gap junction protein beta 1 (Cx32), GJC2 gap junction protein gamma 2 (Cx47), CAV1 Caveolin 1, CAV2 Caveolin 2, OCLN Occludin, DBN1 Drebrin