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.

Bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra-terminal domain (BET) protein family, plays a crucial role in regulating inflammation and oxidative stress that are tightly related to stroke development and progression. Consequently, BRD4 blockade has attracted increasing interest for associated neurological diseases, including stroke. dBET1 is a novel and effective BRD4 degrader through the proteolysis-targeting chimera (PROTAC) strategy. We hypothesized that dBET1 protects against brain damage and neurological deficits in a transient focal ischemic stroke mouse model by reducing inflammation and oxidative stress and preserving the blood–brain barrier (BBB) integrity. Post-ischemic dBET1 treatment starting 4 h after stroke onset significantly ameliorated severe neurological deficits and reduced infarct volume 48 h after stroke. dBET1 markedly reduced inflammation and oxidative stress after stroke, indicated by multiple pro-inflammatory cytokines and chemokines including IL-1β, IL-6, TNF-α, CCL2, CXCL1 and CXCL10, and oxidative damage markers 4-hydroxynonenal (4-HNE) and gp91phox and antioxidative proteins SOD2 and GPx1. Meanwhile, stroke-induced BBB disruption, increased MMP-9 levels, neutrophil infiltration, and increased ICAM-1 were significantly attenuated by dBET1 treatment. Post-ischemic dBET1 administration also attenuated ischemia-induced reactive gliosis in microglia and astrocytes. Overall, these findings demonstrate that BRD4 degradation by dBET1 improves acute stroke outcomes, which is associated with reduced neuroinflammation and oxidative stress and preservation of BBB integrity. This study identifies a novel role of BET proteins in the mechanisms resulting in ischemic brain damage, which can be leveraged to develop novel therapies.

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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. Stroke 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. Mechanistically, adropin neuroprotection depends on endothelial nitric oxide synthase and is associated with reduced BBB permeability and neuroinflammation. We identify adropin as a novel neuroprotective peptide with the potential to improve stroke outcomes.

Introduction: Accumulating evidence indicates that b romodomain and e xtra- t erminal domain (BET) proteins play a pivotal role in the transcriptional regulation of the inflammatory response, and consequently, BET inhibition has emerged as a promising novel therapeutic strategy to reduce inflammation linked to many diseases, including ischemic brain injury. dBET1 is the first BET-targeted proteolysis-targeting chimera (PROTAC) that induces a rapid and efficient deletion of BET proteins in vivo . Hypothesis: In this study, we hypothesized that dBET1 protects against brain damage and neurological deficits in a transient cerebral ischemia mouse model through the regulation of the inflammatory process. Methods: Adult C57BL/6 WT mice were subjected to transient cerebral ischemia surgery (tMCAO) and were administered intraperitoneally with dBET1 (30 mg/kg) or vehicle at 4 and 24 hours after the stroke onset. Infarct volume, neurological deficits, blood-brain barrier integrity, and several inflammatory mediators were examined 48h after tMCAO. Results: The expression level of the BET protein, BRD4, in the cerebral cortex was remarkably lower in the dBET1-treated animals compared to the controls. dBET1 significantly reduced infarct volume (56.2±7.9%, P≤0.01, n=13-14) and improved the neurological performance in the open field test, vertical grid test, and neurological deficit scoring (P≤0.05, n=19-20). In dBET1-treated mice, in both ischemic striatum and cerebral cortex, the inflammation-associated reactive gliosis in astrocytes and microglia was significantly attenuated (P≤0.05, n=6), along with a reduction in stroke-mediated blood-brain barrier damage and neutrophil infiltration. Mechanistically, dBET1 produced a dramatic decrease in the expression levels of pro-inflammatory mediators, while it enhanced levels of anti-inflammatory mediators (P≤0.05, n=6). Conclusion: Overall, this study provides direct in vivo evidence that BET protein degradation with dBET1 suppresses neuroinflammation and leads to anatomical and functional protection against ischemic brain damage. Blocking BET proteins is a promising new strategy to reduce neuroinflammation and eventually improve functional outcomes in stroke.

Background: Receptor-interacting serine-threonine protein kinase 2 (Ripk2) is involved in the signaling pathways of members of the NOD family of cytosolic pattern recognition receptors. Ripk2 has been implicated in the caspase-1 pathway of hypoxia and ischemia-induced neuronal cell death, as well as the pathology of neuroinflammatory diseases like multiple sclerosis. Ischemic stroke is characterized by brain injury resulting from initial hypoxia/ischemia-induced cell death and secondary neuroinflammation. Hypothesis: Since Ripk2 is implicated in both pathways, we hypothesized that the genetic deletion of Ripk2 will improve outcomes for animals after experimental stroke. Methods: We utilized Ripk2 knockout ( Ripk2 -/- ) mice (3-4 months old) and subjected them to 45min of transient middle cerebral artery occlusion (tMCAO) along with wild-type (WT) control littermates. Infarct size was measured at 24h by TTC staining. RNA was isolated from the ipsilateral and contralateral cortices for RT-qPCR. Weight grip test, vertical grid test, and open field behavioral tests were conducted before and up to 21 days after tMCAO with novel object recognition and Y-maze tests occurring at day 28. Results: After 24h, Ripk2 -/- mice had significantly smaller infarct sizes than wild-type (WT) mice. Ripk2 -/- mice also had much lower expression of proinflammatory cytokines ( Tnfα, Il1β, and Il6 ) and chemokines ( Ccl2 and Cxcl1 ) in the ipsilateral cortex relative to WT, implicating a diminished proinflammatory response to ischemia/reperfusion injury in Ripk2 -/- animals. Ripk2 -/- mice displayed significantly better neurological deficit scores compared to WT at 24h, 48h, 7d, 14d, and 21d post-tMCAO. Ripk2 -/- mice also performed much better than WT at acute time points in a battery of behavioral tests, such as the weight grip test, vertical grid test, and open field test. Neurological assessment via novel object recognition and Y-maze tests showed no difference between genotypes at 28d. Conclusions: Our findings support the hypothesis that Ripk2 plays a detrimental role in the pathology of ischemic stroke. Understanding Ripk2’s role in stroke pathology may reveal a novel therapeutic target for the treatment of ischemic stroke.

Background: Adropin is a peptide encoded by the energy homeostasis associated gene ( Enho ) highly expressed in the brain. Our unpublished data revealed robust effects of adropin on reducing neurovascular damage and behavioral impairments in young mice following stroke. However, it is unknown whether these protective effects of adropin are observed in aged animals after stroke. Hypothesis: Considering that aging is the most critical risk factor for stroke, we hypothesized that adropin provides neuroprotection in aged mice subjected to ischemic stroke. Methods: Aged (18-24 months old) male adropin knockout ( Enho -/- ), adropin overexpressing transgenics (AdrTg), and their corresponding control littermates were subjected to permanent middle cerebral artery occlusion (pMCAO). Adhesive removal, nesting behavior, and novel object recognition test were conducted before and up to 14 days after MCAO. Infarct volume was quantified by MRI at 14 days post-MCAO. In a separate experiment, we utilized aged male Enho -/- and AdrTg mice subjected to 30 min of transient MCAO (tMCAO). To investigate the potential therapeutic benefit of adropin in stroke, aged wild-type mice were randomly selected to receive a bolus injection of vehicle or synthetic adropin (900 nmol/kg; i.v.) at the onset of ischemia. Neurobehavioral tests were performed before and at 48h after stroke, and infarct size was measured by TTC staining at 48h. Results: Adropin deficiency consistently increased infarct volume and exacerbated neurological impairments in permanent and transient MCAO mice compared to Enho -/- . In contrast, transgenic overexpression of adropin significantly improved neurological function compared to WT mice. Also, AdrTg mice displayed better recovery of recognition memory function, whereas Enho -/- mice exhibited worse stroke outcomes compared to wild-type littermates following pMCAO. Similarly, treatment with synthetic adropin peptide resulted in a smaller stroke volume and better motor function recovery than vehicle-treated mice following tMCAO. Conclusions: These findings consistently show that transgenic overexpression of adropin or post-ischemic treatment with adropin peptide is neuroprotective in aged mice subjected to ischemic stroke.