Adropin is a highly-conserved peptide that has been shown to preserve endothelial barrier function. Blood-brain barrier (BBB) disruption is a key pathological event in cerebral ischemia. However, the effects of adropin on ischemic stroke outcomes remain unexplored. Hypothesizing that adropin exerts neuroprotective effects by maintaining BBB integrity, we investigated the role of adropin in stroke pathology utilizing loss- and gain-of-function genetic approaches combined with pharmacological treatment with synthetic adropin peptide. Long-term anatomical and functional outcomes were evaluated using histology, MRI, and a battery of sensorimotor and cognitive tests in mice subjected to ischemic stroke. Brain ischemia decreased endogenous adropin levels in the brain and plasma. Adropin treatment or transgenic adropin overexpression robustly reduced brain injury and improved long-term sensorimotor and cognitive function in young and aged mice subjected to ischemic stroke. In contrast, genetic deletion of adropin exacerbated ischemic brain injury, irrespective of sex. Mechanistically, adropin treatment reduced BBB damage, degradation of tight junction proteins, matrix metalloproteinase-9 activity, oxidative stress, and infiltration of neutrophils into the ischemic brain. Adropin significantly increased phosphorylation of endothelial nitric oxide synthase (eNOS), Akt, and ERK1/2. While adropin therapy was remarkably protective in wild-type mice, it failed to reduce brain injury in eNOS-deficient animals, suggesting that eNOS is required for the protective effects of adropin in stroke. These data provide the first causal evidence that adropin exerts neurovascular protection in stroke through an eNOS-dependent mechanism. We identify adropin as a novel neuroprotective peptide with the potential to improve stroke outcomes.

Background: Adropin is a peptide encoded by the energy homeostasis-associated gene ( Enho ) that is highly expressed in the brain. Aging and stroke are associated with reduced adropin levels in the brain and plasma. We showed that treatment with synthetic adropin provides long-lasting neuroprotection in permanent ischemic stroke. However, it is unknown whether the protective effects of adropin are observed in aged animals following cerebral ischemia/reperfusion. We hypothesized that adropin provides neuroprotection in aged mice subjected to transient middle cerebral artery occlusion. Methods: Aged (18–24 months old) male mice were subjected to 30 minutes of middle cerebral artery occlusion followed by 48 hours or 14 days of reperfusion. Sensorimotor (weight grip test and open field) and cognitive tests (Y-maze and novel object recognition) were performed at defined time points. Infarct volume was quantified by 2,3,5-triphenyltetrazolium chloride staining at 48 hours or Cresyl violet staining at 14 days post–middle cerebral artery occlusion. Blood-brain barrier damage, tight junction proteins, and MMP-9 (matrix metalloproteinase-9) were assessed 48 hours after middle cerebral artery occlusion by ELISA and Western blots. Results: Genetic deletion of Enho significantly increased infarct volume and worsened neurological function, whereas overexpression of adropin dramatically reduced stroke volume compared to wild-type controls. Postischemic treatment with synthetic adropin peptide given at the onset of reperfusion markedly reduced infarct volume, brain edema, and significantly improved locomotor function and muscular strength at 48 hours. Delayed adropin treatment (4 hours after the stroke onset) reduced body weight loss, infarct volume, and muscular strength dysfunction, and improved long-term cognitive function. Postischemic adropin treatment significantly reduced blood-brain barrier damage. This effect was associated with reduced MMP-9 and preservation of tight junction proteins by adropin treatment. Conclusions: These data unveil a promising neuroprotective role of adropin in the aged brain after transient ischemic stroke via reducing neurovascular damage. These findings suggest that poststroke adropin therapy is a potential strategy to minimize brain injury and improve functional recovery in ischemic stroke patients.

Background: Receptor-interacting protein kinase 2 (RIPK2) is a serine/threonine kinase whose activity propagates inflammatory signaling through its association with pattern recognition receptors (PRRs) and subsequent TAK1, NF-κB, and MAPK pathway activation. After stroke, dead and dying cells release a host of damage-associated molecular patterns (DAMPs) that activate PRRs and initiate a robust inflammatory response. We hypothesize that RIPK2 plays a damaging role in the progression of stroke injury by enhancing the neuroinflammatory response to stroke and that global genetic deletion or microglia-specific conditional deletion of Ripk2 will be protective following ischemic stroke. Methods: Adult (3–6 months) male mice were subjected to 45 min of transient middle cerebral artery occlusion (tMCAO) followed by 24 h, 48 h, or 28 days of reperfusion. Aged male and female mice (18–24 months) were subjected to permanent ischemic stroke and sacrificed 48 h later. Infarct volumes were calculated using TTC staining (24–48 h) or Cresyl violet staining (28d). Sensorimotor tests (weight grip, vertical grid, and open field) were performed at indicated timepoints. Blood–brain barrier (BBB) damage, tight junction proteins, matrix metalloproteinase-9 (MMP-9), and neuroinflammatory markers were assessed via immunoblotting, ELISA, immunohistochemistry, and RT-qPCR. Differential gene expression profiles were generated through bulk RNA sequencing and nanoString^®. Results: Global genetic deletion of Ripk2 resulted in decreased infarct sizes and reduced neuroinflammatory markers 24 h after stroke compared to wild-type controls. Ripk2 global deletion also improved both acute and long-term behavioral outcomes with powerful effects on reducing infarct volume and mortality at 28d post-stroke. Conditional deletion of microglial Ripk2 (mKO) partially recapitulated our results in global Ripk2 deficient mice, showing reductive effects on infarct volume and improved behavioral outcomes within 48 h of injury. Finally, bulk transcriptomic profiling and nanoString data demonstrated that Ripk2 deficiency in microglia decreases genes associated with MAPK and NF-κB signaling, dampening the neuroinflammatory response after stroke injury by reducing immune cell activation and peripheral immune cell invasion. Conclusions: These results reveal a hitherto unknown role for RIPK2 in the pathogenesis of ischemic stroke injury, with microglia playing a distinct role. This study identifies RIPK2 as a potent propagator of neuroinflammatory signaling, highlighting its potential as a therapeutic target for post-stroke intervention.

Ischemic stroke occurs when a clot forms in the brain vasculature that starves downstream tissue of oxygen and nutrients resulting in cell death. The tissue immediately downstream of the blockage, the core, dies within minutes, but the surrounding tissue, the penumbra is potentially salvageable. Prostaglandin E₂ binds to four different G-protein coupled membrane receptors EP1–EP4 mediating different and sometimes opposing responses. Pharmacological activation of the EP4 receptor has already been established as neuroprotective in stroke, but the mechanism(s) of protection are not well-characterized. In this study, we hypothesized that EP4 receptor activation reduces ischemic brain injury by reducing matrix metalloproteinase (MMP)-3/-9 production and blood-brain barrier (BBB) damage. Rats underwent transient ischemic stroke for 90 min. Animals received an intravenous injection of either the vehicle or L-902,688, a highly specific EP4 agonist, at the onset of reperfusion. Brain tissue was harvested at 24 h. We established a dose-response curve and used the optimal dose that resulted in the greatest infarct reduction to analyze BBB integrity compared to vehicle-treated rats. The presence of IgG, a blood protein, in the brain parenchyma is a marker of BBB damage, and L-902,688 (1 mg/kg; i.v.) dramatically reduced IgG extravasation (P < 0.05). Consistent with these data, we assessed zona occludens-1 and occludin, tight junction proteins integral to the maintenance of the BBB, and found reduced degradation with L-902,688 administration. With immunoblotting, qRT-PCR, and/or a fluorescence resonance energy transfer (FRET)-based activity assay, we next measured MMP-3/-9 since they are key effectors of BBB breakdown in stroke. In the cerebral cortex, not only was MMP-3 activity significantly decreased (P < 0.05), but L-902,688 treatment also reduced MMP-9 mRNA, protein, and enzymatic activity (P < 0.001). In addition, post-ischemic administration of the EP4 agonist significantly reduced pro-inflammatory cytokines IL-1β (P < 0.05) and IL-6 (P < 0.01) in the ischemic cerebral cortex. Most importantly, one injection of L-902,688 (1 mg/kg; i.v) at the onset of reperfusion significantly reduces neurological deficits up to 3 weeks later (P < 0.05). Our data show for the first time that pharmacological activation of EP4 with L-902,688 is neuroprotective in ischemic stroke by reducing MMP-3/-9 and BBB damage.

Background: Matrix metalloproteinases are important factors in the molecular mechanisms leading to neuronal injury in many neurological disorders. Matrix metalloproteinase (MMP)-9 is up-regulated after cerebral ischemia and neuroinflammation and is actively involved in blood–brain barrier disruption. Current methods of measuring MMP-9 activity, such as gelatin-substrate zymography, are unspecific and arduous. Here we developed an immunocapture assay with high efficiency, specificity, and sensitivity for quantifying endogenously active as well as total MMP-9 activity. Results: A fluorescence resonance energy transfer (FRET) peptide-based immunocapture assay was developed that enables the accurate assessment of total and active forms of MMP-9 in complex biological samples. The FRET assay demonstrated correct and efficient binding of MMP-9 to a mouse monoclonal MMP-9 antibody and high specificity of the immunocapture antibody for MMP-9. Total and active levels of MMP-9 were measured in rat brain homogenates, plasma, human HT-1080 conditioned media, and RBE4 endothelial cell lysates. The FRET immunocapture assay yielded highly similar results for total MMP-9 activity when compared to gelatin-substrate zymography. Conclusions: We suggest that the new FRET peptide-based immunocapture assay is a viable replacement of zymography for sensitive and high throughput quantification of MMP-9 activity in biological samples.