Abstract
Rempe RG, Hartz AMS, Soldner ELB, Sokola BS, Alluri SR, Abner EL, Kryscio RJ, Pekcec A, Schlichtiger J, Bauer B. J Neurosci 2018;38:4301–4315
The blood-brain barrier is dysfunctional in epilepsy, thereby contributing to seizure genesis and resistance to antiseizure drugs. Previously, several groups reported that seizures increase brain glutamate levels, which leads to barrier dysfunction. One critical component of barrier dysfunction is brain capillary leakage. Based on our preliminary data, we hypothesized that glutamate released during seizures mediates an increase in matrix-metalloproteinase (MMP) expression and activity levels, thereby contributing to barrier leakage. To test this hypothesis, we exposed isolated brain capillaries from male Sprague Dawley rats to glutamate ex vivo and used an in vivo/ex vivo approach of isolated brain capillaries from female Wistar rats that experienced status epilepticus as an acute seizure model. We found that exposing isolated rat brain capillaries to glutamate increased MMP-2 and MMP-9 protein and activity levels, and decreased tight junction protein levels, which resulted in barrier leakage. We confirmed these findings in vivo in rats after status epilepticus and in brain capillaries from male mice lacking cytosolic phospholipase A2 Together, our data support the hypothesis that glutamate released during seizures signals an increase in MMP-2 and MMP-9 protein expression and activity levels, resulting in blood-brain barrier leakage. SIGNIFICANCE STATEMENT: The mechanism leading to seizure-mediated blood-brain barrier dysfunction in epilepsy is poorly understood. In the present study, we focused on defining this mechanism in the brain capillary endothelium. We demonstrate that seizures trigger a pathway that involves glutamate signaling through cytosolic phospholipase A2, which increases MMP levels and decreases tight junction protein expression levels, resulting in barrier leakage. These findings may provide potential therapeutic avenues within the blood-brain barrier to limit barrier dysfunction in epilepsy and decrease seizure burden.
Commentary
The very first documented evidence that the brain microenvironment is protected from free exchange with the bloodstream came from experiments conducted by physician and scientist Paul Ehrlich in 1885. Ehrlich observed that intravenous injection of a dye in experimental animals does not stain all organs evenly but instead leaves the brain unstained. It is now well-known that an intact blood–brain barrier is essential for normal brain function and that head injuries or certain neurological diseases can compromise this barrier, thereby worsening the condition (1). In epilepsy, a leaking blood–brain barrier is believed to contribute to epileptogenesis after status epilepticus and in chronic epilepsy. Repairing this leaky barrier in epilepsy has since long been sought after as a potential therapeutic strategy. To prevent brain barrier leakage or restore its function, it is important to fully understand the underlying mechanisms. The study of Rempe and colleagues provides an important step toward this goal: Using an elegant ex vivo preparation of brain capillaries, not only did they elucidate some of the signaling pathways and enzymes involved in making a blood–brain barrier leaky but also identified potential pharmacological treatment targets.
The blood–brain barrier is maintained by the neurovascular unit, which is comprised of endothelial cells forming the wall of the brain capillaries (the actual physical barrier) as well as closely adjacent pericytes, astrocytes, and neurons (2). Tight junctions connect the endothelial cells and are essential for preserving the blood–brain barrier. They are formed by tight junction proteins that are downregulated in conditions of blood–brain barrier leakage. Their expression is regulated by matrix metalloproteinases (MMP), a family of protein-degrading enzymes that break down the extracellular matrix and have been previously implicated in brain–blood barrier leakage. MMPs are upregulated in epilepsy, but the mechanisms of how they are activated and their direct functions in brain capillaries have been unclear. Importantly, before this study of Rempe et al., not much was known about how their activation after seizure could be pharmacologically limited as a potential treatment for epilepsy.
Rempe and colleagues have used an ex vivo preparation of brain capillaries to reveal pharmacologically targetable mechanisms underlying brain barrier leakage. This elegant approach allowed them to assess the effects of acute seizures on barrier function and, at the same time, provided them with an easily manipulatable system to test potential treatment options. Using this system, they showed that the matrix metalloproteinases MMP-2 and MMP-9 are expressed in low levels in healthy brain capillaries but strongly upregulated after pilocarpine-induced status epilepticus. Importantly, they showed that MMPs are enzymatically active in brain capillaries and that this enzymatic activity is likewise upregulated and accompanied by brain–blood barrier leakage after status epilepticus. These changes were only observed when rats experienced status epilepticus but not by pilocarpine alone, corroborating that this is a general phenomenon of status epilepticus and not an effect of the drug.
Making use of the fact that glutamate is released in excess during seizures, the authors created an “epileptic brain barrier in a dish.” They showed that exposure of brain capillary preparations to glutamate induced barrier leakage and increased MMP-2 and -9—similar to what is observed in vivo in the brain after status epilepticus. Using this simplified in vitro model of seizure-induced blood–brain barrier leakage, the authors were able to reveal the molecular signaling pathways responsible for MMP upregulation. They showed that NMDA receptors were necessary for both the increase in MMPs and the barrier leakage induced by excessive glutamate and that this effect was mediated through the cytosolic phospholipase A2 (cPLA2). As an important validation of their in vitro findings, Rempe et al. used genetic and pharmacological approaches to show that the same molecular signaling cascade leads to MMP upregulation and blood–brain barrier leakage in vivo as well.
cPLA2 is of specific interest in epilepsy because it is upregulated in several animal models of seizure and epilepsy (e.g., [3]). Moreover, its function to hydrolyze phospholipids to arachidonic acid, a precursor of prostaglandin, suggests a potential treatment target to reduce neuroinflammation in epilepsy (4). Therefore, cPLA2 inhibition may be involved in multiple disease mechanisms in epilepsy in addition to blood–brain barrier leakage mediated by increased MMPs. Indeed, there are a few findings that support cPLA2 as a target to ameliorate epilepsy and other neurological diseases. For example, the compounds of Centella asiatica, a plant used in traditional medicine for several neurological disorders, which also has anticonvulsant properties, was shown to inhibit cPLA2 (5, 6). Moreover, a change in membrane lipid metabolism mediated by cPLA2 may contribute to the anticonvulsant effect of the ketogenic diet (7). It remains to be seen whether or not cPLA2 plays a role in altering the blood–brain barrier integrity in either of these treatments.
In summary, these findings make a strong case that cPLA2 could be a therapeutic target in epilepsy. However, the fact that cPLA2 has many functions in the cell may not be only an advantage: The risk of unwanted side effects increases when the therapeutic strategy is targeted at a molecule upstream of the actual disease mechanism. This raises the question whether an approach directly inhibiting MMPs could be more specific and thus promising in epilepsy. The authors addressed this question by showing that broad spectrum inhibition of MMPs prevents blood–brain barrier leakage, but they did not further assess which MMP may make the strongest contribution. MMP-9 is an interesting candidate because of availability of an FDA-approved inhibitor (the antibiotic minocycline), which has shown anticonvulsant properties (8) and because of its involvement in other neurological diseases, such as autism. In the autism spectrum disorder fragile X syndrome, for example, minocycline rescues phenotypes in the mouse model and improved behavior in patients in an open-label study (9). Notably, the blood–brain barrier was shown to be compromised in autism, and minocycline has ameliorated blood–brain barrier damage in a rat model of autism (10). It is tempting to speculate that overlapping mechanisms damaging the blood–brain barrier, possibly through MMP-9, may contribute to the higher incidence of epilepsy in autism.
While there is probably not one single “leaker” responsible for the compromised blood–brain barrier in epilepsy, the findings of Rempe et al. (taken together with previous studies) strongly suggest a crucial role for MMP-9 and other matrix metalloproteinases. Future studies are needed to identify the optimal route to pharmacologically target this mechanism. This could be direct inhibition of MMP-9 (which has shown some promise in epilepsy and autism), inhibition of other MMPs, such as MMP-2 (less well studied in this context), or pharmacological intervention one step above at the level of the MMP activator cPLA2, possibly able to correct two disease mechanisms, altered MMP activity and elevated neuroinflammation.