TNFα disrupts blood brain barrier integrity to maintain prolonged depressive-like behavior in mice

Highlights

• We describe a prolonged (>4 weeks) learned helplessness (LH) depression model in mice.

• Prolonged LH was associated with elevated hippocampal TNFα, IL-17A and IL-23 levels.

• Blood-brain barrier (BBB) disruption was evident in mice with prolonged LH.

• Prolonged LH and elevated cytokines were rapidly reversed by Fingolimod or TNFα inhibitor treatment.

• TNFα-mediated disruption of the BBB may contribute to impaired recovery from LH.

Abstract

Recovery from major depressive disorder is difficult, particularly in patients who are refractory to antidepressant treatments. To examine factors that regulate recovery, we developed a prolonged learned helplessness depression model in mice. After the induction of learned helplessness, mice were separated into groups that recovered or did not recover within 4 weeks. Comparisons were made between groups in hippocampal proteins, inflammatory cytokines, and blood brain barrier (BBB) permeability. Compared with mice that recovered and control mice, non-recovered mice displaying prolonged learned helplessness had greater hippocampal activation of glycogen synthase kinase-3 (GSK3), higher levels of tumor necrosis factor-α (TNFα), interleukin-17A, and interleukin-23, increased permeability of the blood brain barrier (BBB), and lower levels of the BBB tight junction proteins occludin, ZO1, and claudin-5. Treatment with the GSK3 inhibitor TDZD-8 reduced inflammatory cytokine levels, increased tight junction protein levels, and reversed impaired recovery from learned helplessness, demonstrating that prolonged learned helplessness is reversible and is maintained by abnormally active GSK3. In non-recovered mice with prolonged learned helpless, stimulation of sphingosine 1-phosphate receptors by Fingolimod or administration of the TNFα inhibitor etanercept repaired the BBB and reversed impaired recovery from prolonged learned helplessness. Thus, disrupted BBB integrity mediated in part by TNFα contributes to blocking recovery from prolonged learned helplessness depression-like behavior. Overall, this report describes a new model of prolonged depression-like behavior and demonstrates that stress-induced GSK3 activation contributes to disruption of BBB integrity mediated by inflammation, particularly TNFα, which contributes to impaired recovery from prolonged learned helplessness.

Introduction

Major depressive disorder, which afflicts nearly 17% of people in the United States, is defined by the presence of multiple debilitating symptoms and by their prolonged duration (Kessler et al., 2005). Stress is a common precipitating factor of depression, and stress is often used to induce rodent models of depression (Kessler, 1997, Pariante and Lightman, 2008). These rodent models are evaluated by depression-like behaviors because rodents are not thought to experience the equivalent of major depressive disorder (Gould and Gottesman, 2006, Nestler and Hyman, 2010).

One robust rodent depression model is learned helplessness, in which rodents are exposed to uncontrollable and inescapable mild foot shocks as the aversive stimuli (Chourbaji et al., 2005, Maier and Seligman, 2016, Vollmayr and Gass, 2013). Upon subsequent exposure to foot shocks with escape freely available, rodents that display learned helplessness fail to leave the foot shock chamber. Learned helplessness is considered a behavioral model of depression, particularly involving despair and a coping deficit in response to stress, because it is accompanied by changes typical of depression, including decreased brain regional levels of serotonin, norepinephrine, dopamine, and brain-derived neurotrophic factor (BDNF), decreased home cage activity, elevated corticosteroid levels, increased rapid eye movement during sleep, and displays of other depression-like behaviors, such as impaired social interactions, anhedonia, and increased immobility in the tail suspension and forced swim tests (Chourbaji et al., 2005, Henn and Vollmayr, 2005, Vollmayr and Gass, 2013). Furthermore, susceptibility to learned helplessness is diminished by treatment with antidepressants and other drugs, such as a high dose of lithium (Beurel et al., 2011, Vollmayr and Gass, 2013). Lithium is an inhibitor of glycogen synthase kinase-3 (GSK3), which refers to two isoforms that are predominantly controlled by inhibitory phosphorylation on serine21-GSK3α and serine9-GSK3β (Beurel et al., 2015). Substantial evidence indicates that abnormally active GSK3 promotes depression and animal models of depression, which may derive in part from the promotion of inflammation by GSK3 (Jope, 2011).

Numerous studies have reported treatments and mechanisms that regulate susceptibility to onset of depression-like behaviors in rodents (Gould and Gottesman, 2006, Nestler and Hyman, 2010), but less is known about treatments and mechanisms that control recovery from prolonged depression-like behaviors. We observed that some mice display prolonged learned helplessness, rather than recovering in the usual 2–4 week period. This led us to examine mechanisms that may contribute to impaired recovery. Inflammatory cytokines are elevated in depressed patients, including treatment refractory patients. One of the possible mechanisms whereby cytokines can maintain depressive symptoms is by affecting the blood brain barrier (BBB). There is evidence of impaired integrity of the BBB in depressed patients (Lavoie et al., 2010, Najjar et al., 2013) and rodent models of depression (Esposito et al., 2001, Friedman et al., 1996, Santha et al., 2015, Sharma et al., 1995), and inflammatory cytokines, such as tumor necrosis factor-α (TNFα), can impair BBB integrity (Rochfort and Cummins, 2015). Endothelial cells connected by tight junctions (TJs) constitute an integral component of the BBB. TJs are comprised of occludin, claudin, ZO-1, ZO-2 and other junction adhesion molecules that maintain structural and functional integrity of the brain endothelium (Obermeier et al., 2013). GSK3 inhibition promotes TJ stability in brain endothelial cells by increasing the levels of occludin and claudin-5 (Ramirez et al., 2013), suggesting GSK3 inhibition may protect the BBB by stabilizing TJ proteins. Decreased levels of TJ proteins and BBB breakdown occur after trauma, stroke, multiple sclerosis, and Alzheimer’s disease, conditions associated with increased activated GSK3, neuroinflammation and increased prevalence of depression.

Therefore, we examined links between recovery from prolonged learned helplessness and GSK3, inflammation and BBB integrity. The results show that BBB permeability is increased in mice that do not recover from prolonged learned helplessness, and that recovery and BBB integrity can be improved pharmacologically by treatment with a GSK3 inhibitor, Fingolimod, or anti-TNFα. These results indicate that stress-induced GSK3 activation promotes inflammation that contributes to disruption of BBB integrity, and this is associated with impaired recovery from prolonged learned helplessness.