Only a handful of studies have explored BBB sex differences, most indirectly and in vitro37 but, to our knowledge, none did so in the context of chronic stress in mice or MDD. Overall, our findings indicate that chronic social and subchronic variable stressors alter BBB integrity in the mouse female brain through loss of the tight junction protein Cldn5 in the PFC and, to some extent, other mood-related brain regions such as the NAc. Importantly, these vascular alterations are also present in postmortem human brain samples from women with MDD. In mice, viral-mediated downregulation of Cldn5 in the PFC is sufficient to promote anxiety- and depression-like behaviours including social avoidance, anhedonia, and helplessness supporting a causal role in the establishment of maladaptive stress responses and possibly, mood disorders. We did not observe stress-induced BBB dysfunction in the male PFC7 indicating that chronic stress and depression affect the neurovasculature in a sex-specific manner. Different stress paradigms elicit specific anxiety- and depression-like behaviours according to sex, each recapitulating certain aspects of the symptoms and molecular features of MDD38.
A potential limitation of the female CSDS model we have used here is exposure to a male stressor (i.e., antagonistic social confrontations by a larger male aggressor). In male C57BL/6, the CSDS paradigm has been shown to have relevant etiological, predictive and face validity14. However, additional studies are warranted to confirm that this model is also etiologically relevant for female mice. Furthermore, SI tests were conducted using a male social target and we cannot exclude a possible confounding effect of intersex social interaction on the SI ratio values. Performing these tests with a female target may result in a different stratification of SS and RES mice, as we report for our virus-injected cohorts. We chose to use male mice as social targets in other contexts because social stress was performed by male aggressors19. Physical injuries are always a concern when running the CSDS paradigm either in males or females. Indeed, sickness behaviours could account for behavioral changes through stimulation of the immune system and entry of inflammatory mediators into the brain via altered BBB permeability. Therefore, physical examinations of animals were performed and no difference in the number of wounds was observed (Supplementary Figs. 1a and 8b) suggesting that susceptible vs resilient behavioral phenotypes were not due to physical injury. Finally, stress resilience is a fluid concept, and it is still debated whether or not it can be defined as a trait39. The complex neurobiological interactions underlying SS and RES phenotypes are not fully understood and are highly context-dependent. Coping strategies (i.e., social interactions) may be considered adaptative in some circumstances and maladaptive in others, and such mechanisms have been poorly investigated in females40. Thus, further studies are needed to better understand individual differences in stress-induced resilience vs vulnerability and how these behavioural responses and underlying mechanisms relate to human mood disorders.
Only females are susceptible to 6-d SCVS; however, both sexes display depression-like behaviours after weeks of stress exposure9, 15 or if behavioural testing is performed 30 days after the 6-d SCVS paradigm41. Discrepancies in these behaviours could explain the sex-specific regional vascular effects observed and, possibly, sex differences associated with MDD. Magnetic resonance imaging (MRI) studies also support this idea, with unmedicated women with MDD showing decreased grey matter volume in limbic regions, including the left ventral PFC, while this reduction is observed in striatal regions for men with MDD42. Our study is also in line with recent clinical observations reporting region-specific BBB disruption in psychiatric disorders43, 44, although sex differences were not addressed.
Our work not only highlights fundamental sex differences in stress-induced neurovascular responses but also provides mechanistic insights by identifying key pathways and genes involved. Bulk tissue sequencing studies show a major rearrangement of transcriptional patterns in mood-related brain regions in MDD with low overlap between men vs women with MDD (~10%)9, 11. These marked sex differences are also observed in mouse models of depression9, 15. Neuronal contribution is undeniable, notably via changes in neurotransmitter systems9. Nevertheless, a significant enrichment for endothelium-related genes is also present9 but had never been explored. Bulk RNA sequencing revealed a similar enrichment for genes related to this cell population in schizophrenia45, reinforcing the involvement of the neurovasculature in psychiatric disorders. Although the resilience phenotype in female mice is not as clearly defined as it is for their male counterparts when analysing behaviours19, 24, our endothelium transcriptomic profiling revealed a distinct resilience-associated pattern when compared to animals displaying anxiety- or depression-like behaviours induced by either chronic social or subchronic variable stress. We found secreted enzyme phospholipase A2 Group VII (Pla2g7) as a gene significantly upregulated in SS vs RES mice, and Pla2g7 methylation is associated with increased risk of coronary heart disease specifically in women46. On the other hand, carnitine acyl carnine translocase (Slc25a20) is decreased in the PFC of SS females when compared to RES, which is consistent with previous studies reporting decreased acetyl-l-carnitine in MDD patients47.
Interestingly, both sexes show significant endothelium gene expression changes in the omega-3/omega-6 fatty acid synthesis pathways in the PFC following chronic social stress. However, these alterations were observed in RES vs CTRL for females and between SS vs CTRL in males. This pathway has been extensively studied in human depression as well as in animal models of mood disorders with omega-3 deficiency linked to neuronal atrophy in the medial PFC of male mice, concomitant with anxiety- and depressive-like behaviours48. Fatty acids play an important role in the regulation of systemic49 and endothelium inflammation50, thus providing an intriguing association between depression-related disruptions in the neuroimmune axis51 and BBB hyperpermeability. Similarities between the PFC female resilient and male susceptible endothelial transcriptome profiles for this pathway and the fact that both subgroups maintain BBB integrity in this brain region despite chronic stress exposure suggest that it could play a protective role in stress-induced BBB permeability loss. With previous reports linking omega-3 fatty acid consumption with resilience to stress in rodents52 and humans53, further studies are warranted to confirm their potential beneficial effect on the neurovasculature and gain mechanistic insights.
A possible mechanism for this sex- and region-specific vulnerability of the BBB is the presence of estrogen receptors on the neurovasculature. Endothelial cells express low levels of functional estrogen receptors54 and estrogen-coupled receptors can enter the cell nucleus and bind to estrogen-responsive elements (ERE) on specific DNA sequences55. Interestingly, ERE and stimulating protein 1 (Sp1) transcription factors were identified on the mouse Cldn5 gene promoter, which allows estrogen receptors to modulate Cldn5 transcription through cooperative interactions of Er/Sp1 with ERE/Sp1 elements56. While high levels of estrogen render female rats more sensitive to stress-induced PFC dysfunction57, this was found to be protective in the striatum58, providing mechanisms to explore for future studies. Our group has recently shown that permissive epigenetic regulation of Cldn5 expression paired with low endothelium Cldn5-repressive transcription factor forkhead box protein O1 is associated with stress resilience in the NAc of male mice, while increased histone deacetylase 1 level and activity is a mediator of stress susceptibility8. Thus, investigating sex-specific epigenetic vascular mechanisms underlying susceptibility vs resilience to stress and BBB hyperpermeability will be important in the future.
Additionally, estrogen-activated receptors have been shown to inhibit the proinflammatory transcription factor NfkB (nuclear factor kappa light chain enhancer of activated B cells), a known regulator of ICAM-1 and E-selectin59. Increased circulating proinflammatory cytokines and NFkB were reported in adolescent MDD and bipolar disorder, correlated with depressive symptoms severity60, and we have identified the proinflammatory NFkB pathway as a mediator of stress susceptibility in NAc endothelial cells of male8 but not female mice (Figs. 4 and 5). Accordingly, we observed a sex-specific increase in circulating sICAM-1 levels only in SS male mice vs pre-CSDS at baseline (Supplementary Fig. 10) and Icam1 levels are increased in the NAc of SS male mice8 but seem to be reduced in the PFC of SS female mice (Supplementary Fig. 10), reinforcing the idea of sex-specific regulatory mechanisms of BBB integrity, possibly through estrogen-mediated pathways. Social defeat stress increases the expression of adhesion molecules in the male mouse brain, including Icam-1 and Sele the gene encoding for E-selectin61. We did observe a trend, that did not reach significance, for higher expression of Sele in the PFC of stressed mice (Supplementary Fig. 10) suggesting that the increase in circulating sE-selectin measured in the blood serum of SS females may come, at least partly, from other brain regions or non-CNS sources. Moreover, elevated blood sE-selectin level was reported in the elderly with mild cognitive impairment and depressive mood62; however, these studies did not address sex differences. Assessment of BBB leakage using MRI scans in patients suffering from bipolar disorder allowed identification of a subpopulation of patients characterized by worse symptoms including the severity of depression, anxiety, chronicity of illness and decreased global functioning63. Despite being commonly used worldwide, it would be unrealistic to apply BBB imaging to a large population scale or in a preventive context highlighting the importance in discovering biomarkers of psychiatric diseases as we aimed to do here. It could be particularly relevant for conditions involving exacerbated inflammation and/or vascular dysfunction and for which MDD prevalence is higher than the general population; for example, stroke or Alzheimer’s disease1.
Many unanswered questions persist regarding BBB adaptations in both health and disease18. Our multidisciplinary approach allowed us to identify sex-specific circulating vascular potential biomarkers as well as candidate genes and pathways that could be relevant to inform on MDD diagnosis and develop treatments. Targeting and regulating tight junction protein integrity at the BBB level could represent an innovative strategy to treat mood disorders43. However, thinking beyond endothelial cells will be important to better understand the complex biology underlying BBB hyperpermeability in MDD. Single-cell sequencing of postmortem brain tissue from individuals with MDD shows important dysfunction in the PFC pyramidal neurons and oligodendrocyte-lineage cells64, but to our knowledge, this had never been investigated for endothelial cells, smooth muscle cells or pericytes. By characterizing sex- and region-specific neurovascular alterations underlying stress susceptibility in mice and human depression we provide valuable clues and highlight the need to consider sex as a biological variable while defining the role of brain barriers in psychiatric diseases. These findings are also important in the context of cardiovascular diseases (CVD), with evidence supporting a bidirectional relationship between depression and CVD, where depression is a predictor for the development of CVD, and vice versa65. Like in MDD, sex differences in age of onset, symptomatology and treatment response exist in CVD, highlighting the possibility of common etiological basis. Thus, investigating the candidate genes and circulating vascular potential biomarkers associated with depression we report here in populations of CVD patients would be highly relevant and could provide clues into shared underlying mechanisms of CVD and MDD pathologies. As an example, we observed an increase in sPAI-1, a prothrombotic plasma protein secreted by endothelial tissue, following social and subchronic variable stress in female, but not male, mice (Fig. 6c, d, Supplementary Fig. 10). Dysfunction of this pathway has been proposed as a link between MDD and CVD66 but underlying mechanisms remain to be elucidated. Genetic variants of the PAI-1 gene (SERPINE1) have been associated with MDD67. An elevated level of PAI-1 proteins is observed in the serum of individuals with major depression disorder68, but is also linked with increased risk of ischemic cardiovascular events such as thrombosis and atherosclerosis69. Antidepressant treatment decreases PAI-1 expression in the brain of male rats67. However, discrepancies exist with PAI-1 deficiency predisposing male mice to depression-like behaviours and resistance to commonly prescribed selective serotonin reuptake inhibitors70. Our findings suggest that PAI-1 may be even more relevant for female stress responses, and it was hypothesized that it could play a role in perinatal depression71, raising interest for future studies on this target to gain mechanistic insights particularly for female rodents and women with MDD.
To sum up, our study shows that chronic stress can induce BBB alterations in the female PFC promoting anxiety, depression-like behaviours, and BBB leakiness (Supplementary Fig. 11). Importantly, loss of tight junction CLDN5 expression was confirmed in postmortem brains samples from women with major depressive disorder supporting relevance for human MDD. These alterations were associated with changes in circulating vascular potential biomarkers in the mouse and human blood serum that will have to be explored in future clinical studies and larger human cohorts to confirm translational value. Conversely, despite chronic stress exposure, BBB integrity is maintained in the PFC of RES animals and this could be related to transcriptomic adaptations and activation of protective signalling pathways in the endothelium (Supplementary Fig. 11). Although this project explored neurovasculature-related changes in two brain regions, many other areas involved in emotion regulation remain unexplored and we hypothesize that stress-induced BBB changes go beyond the NAc and PFC. It will also be intriguing to investigate if age-related neurovascular changes such as BBB breakdown, which has been linked to human cognitive dysfunction72, play a causal role in late-life depression73 which is more prevalent in women74.
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