Social stress induces neurovascular pathology promoting depression

Abstract

Studies suggest that heightened peripheral inflammation contributes to the pathogenesis of major depressive disorder. We investigated the effect of chronic social defeat stress, a mouse model of depression, on blood-brain barrier (BBB) permeability and infiltration of peripheral immune signals. We found reduced expression of the endothelial cell tight junction protein claudin-5 (Cldn5) and abnormal blood vessel morphology in nucleus accumbens (NAc) of stress-susceptible but not resilient mice. CLDN5 expression was also decreased in NAc of depressed patients. Cldn5 downregulation was sufficient to induce depression-like behaviors following subthreshold social stress whereas chronic antidepressant treatment rescued Cldn5 loss and promoted resilience. Reduced BBB integrity in NAc of stress-susceptible or mice injected with adeno-associated virus expressing shRNA against Cldn5 caused infiltration of the peripheral cytokine interleukin-6 (IL-6) into brain parenchyma and subsequent expression of depression-like behaviors. These findings suggest that chronic social stress alters BBB integrity through loss of tight junction protein Cldn5, promoting peripheral IL-6 passage across the BBB and depression.

Figure 1. Social stress vulnerability and MDD is associated with reduced cldn5 expression

A) Experimental timeline of 10-day social defeat stress (CSDS), social interaction (SI) behavioral screening and tissue collection of nucleus accumbens (NAc). B) Quantitative PCR revealed significant changes in the NAc of stress-susceptible (SS) and resilient (RES) mice when compared to unstressed controls (CTRL) for gene expression related to endothelial cell biology, angiogenesis, tight junctions, blood-brain barrier (BBB) formation and cytokines/chemokines. The range of color indicates individual differences within a group – standard error of the mean (SEM) from the average represented by the dashed line. C) Several patterns of gene expression correlated with SI ratio (D). Representative heat maps of SI test are shown on the right. Cldn5 mRNA (E) and protein (F, G) levels were lower in the NAc of SS mice and correlated with social avoidance. Scale bar is set at 20 µm. H) Chronic treatment with imipramine reversed social avoidance and rescued cldn5 loss in SS mice. I) Transmission electron microscopy revealed discontinuity in tight junctions of SS (red arrows), but not resilient, mice. Scale bar is set at 500 nm (250 nm for insets), 52–66 tight junctions/mouse and three mice/group. J) CLDN5 mRNA level was significantly lower in the NAc of MDD patients from two different cohorts. Data represent mean ± SEM, number of animals or patients (n) is indicated on graphs. Two-way ANOVA followed by Bonferroni’s multiple comparison test for antidepressant treatment, correlations were evaluated with Pearson’s correlation coefficient, and one-way ANOVA followed by Bonferroni’s multiple comparison test for other graphs, *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2. Conditional knockdown of cldn5 expression in the NAc is sufficient to induce depression-like behaviors

A) Experimental timeline of NAc bilateral injection of AAV-shRNA or AAV-shRNA-cldn5 viruses and behavioral studies. Cldn5 mRNA (B) and protein (C) levels are reduced following AAV-shRNA-cldn5 injection in the NAc of stress-naïve mice when compared to AAV-shRNA-injected animals. Co-staining with CD31 confirmed lower cldn5 protein level in blood vessels following AAV-shRNA-cldn5 virus injection and doxycycline (Dox) treatment (C). Scale bar is set at 2 µm. Downregulation of cldn5 expression prior to subthreshold micro-defeat (stressed) induced depression-like behaviors as assessed by splash (D), sucrose preference (E), forced swim (F) and social interaction (G) tests. The effect was significant when compared to unstressed mice and stressed AAV-shRNA. H) Experimental timeline of cldn5 rescue experiment. I) Stressed AAV-shRNA-cldn5 mice displayed depression-like behaviors when subjected to doxycycline treatment as assessed with forced swim test and sucrose preference. J) No difference was observed between groups for sucrose preference and social interaction when doxycycline was removed from the water, allowing normal cldn5 expression. Data represent mean ± SEM, number of animals (n) is indicated on graphs. Unpaired t-test for virus validation or two-way ANOVA followed by Bonferroni’s multiple comparison test for behaviors, *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3. Social stress vulnerability is associated with increased blood-brain barrier (BBB) permeability

A) Experimental timeline of 10-day CSDS, SI screening, and magnetic resonance imaging (MRI) scans. Pictures display baseline MRI scan compared to 25 minutes after injection of the gadolinium-contrasting agent (Gd-DTPA). B) Higher Gd-DTPA signal was detected in different brain regions, including the NAc in SS mice and negatively correlated with SI ratio (C). The range of color indicates individual differences within a group - SEM from the average represented by the dashed line. Assessment of BBB permeability with cadaverine Alexa Fluor-555 and Evans blue (EB) dyes revealed significant BBB leakiness in the NAc of SS mice (D, E). EB dye is present in all vessels within 10 min of injection and prior to perfusion (E, left) but can only be detected in the perivascular space of NAc blood vessels in SS mice after circulating 16 hours followed by 5-min perfusion (E, right). Scale bar is set at 20 µm. F, G) Downregulation of cldn5 expression increased BBB permeability to cadaverine Alexa Fluor-555 and EB dyes (H) in AAV-shRNA-cldn5 mice. I) Transmission electron microscopy, conducted 2 hours after injection of horseradish peroxidase (HRP) followed by 20-min perfusion, confirmed increased BBB permeability in the NAc of SS mice with HRP detected within the endothelial cell lining (red arrows). Quantification was performed on 34–61 blood vessels/mouse in three mice/group. Scale bar is set at 500 nm. Data represent mean ± SEM, number of animals (n) is indicated on graphs. Correlations were evaluated with Pearson’s correlation coefficient, two-way ANOVA followed by Bonferroni’s multiple comparison test for BBB permeability in virus-injected mice, one-way ANOVA followed by Bonferroni’s multiple comparison test for other graphs, *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4. Chronic social stress induces peripheral monocyte accumulation and IL-6 passage into the NAc

A) Experimental timeline of 10-day CSDS, SI behavioral screening and tissue collection of ccr2^RFP∷ cx3cr1^GFP mice. B) Stressed ccr2^RFP∷ cx3cr1^GFP mice showed decreased SI scores following 10-day CSDS. Representative heat maps are shown on the right. C) Flow cytometric analysis of forebrain ccr2^RFP+ and cx3cr1^GFP+ cells revealed higher level of ccr2⁺ peripheral monocyte accumulation into the brain of stressed mice. Cells were gated on live CD11b⁺F4/80⁺ (left). Numbers adjacent to gates represent percentages of ccr2⁺cx3cr1 cells among CD11b⁺F4/80⁺ cells. Right panel shows CD45 expression of F4/80⁺ccr2⁺ monocytes and F4/80⁺gfp⁺ microglia. Bar graph shows percentage of ccr2⁺cx3cr1 cells among forebrain CD11b⁺F4/80+ cells. D) IL-6 protein level is increased in the blood and NAc of SS mice after 10-day CSDS. E) Dose response IL-6 i.p. injection did not change IL-6 protein level in the NAc of stress-naïve mice. F) Direct infusion of IL-6 into the NAc (5 min) induces social avoidance when subthreshold micro-defeat was conducted 20 min after the end of the infusion. G) Experimental timeline of peripheral biotinylated-IL6 injection after 10-day CSDS. IL-6-biotin-Neuravidin-Oregon 488 was detectable in the NAc parenchyma of SS mice only after 2h circulation and 5-min perfusion with PBS. H) Biotinylated IL-6 was also detectable in the NAc of AAV-shRNA-cldn5-injected mice. Data represent mean ± SEM, number of animals (n) is indicated on graphs. T-test for CX3CR1-GFP/CCR2-RFP mouse data, one-way ANOVA followed by Bonferroni’s multiple comparison test for IL-6 protein levels and two-way ANOVA followed by Bonferroni’s multiple comparison test for SI ratio after IL-6 or saline infusion, *p < 0.05; **p < 0.01; ***p < 0.001.