Receptor-interacting protein kinase 2 (RIPK2) profoundly contributes to post-stroke neuroinflammation and behavioral deficits with microglia as unique perpetrators.

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.

Neuroprotection by the cannabidiol aminoquinone VCE-004.8 in experimental ischemic stroke in mice.

Synthetic cannabidiol (CBD) derivative VCE-004.8 is a peroxisome proliferator-activated receptor gamma (PPARγ) and cannabinoid receptor type 2 (CB2) dual agonist with hypoxia mimetic activity. The oral formulation of VCE-004.8, termed EHP-101, possesses anti-inflammatory properties and is currently in phase 2 clinical trials for relapsing forms of multiple sclerosis. The activation of PPARγ or CB2 receptors exerts neuroprotective effects by dampening neuroinflammation in ischemic stroke models. However, the effect of a dual PPARγ/CB2 agonist in ischemic stroke models is not known. Here, we demonstrate that treatment with VCE-004.8 confers neuroprotection in young mice subjected to cerebral ischemia. Male C57BL/6J mice, aged 3-4 months, were subjected to 30-min transient middle cerebral artery occlusion (MCAO). We evaluated the effect of intraperitoneal VCE-004.8 treatment (10 or 20 mg/kg) either at the onset of reperfusion or 4h or 6h after the reperfusion. Seventy-two hours after ischemia, animals were subjected to behavioral tests. Immediately after the tests, animals were perfused, and brains were collected for histology and PCR analysis. Treatment with VCE-004.8 either at the onset or 4h after reperfusion significantly reduced infarct volume and improved behavioral outcomes. A trend toward reduction in stroke injury was observed in animals receiving the drug starting 6h after recirculation. VCE-004.8 significantly reduced the expression of pro-inflammatory cytokines and chemokines involved in BBB breakdown. Mice receiving VCE-004.8 had significantly lower levels of extravasated IgG in the brain parenchyma, indicating protection against stroke-induced BBB disruption. Lower levels of active matrix metalloproteinase-9 were found in the brain of drug-treated animals. Our data show that VCE-004.8 is a promising drug candidate for treating ischemic brain injury. Since VCE-004.8 has been shown to be safe in the clinical setting, the possibility of repurposing its use as a delayed treatment option for ischemic stroke adds substantial translational value to our findings.

Targeted BRD4 protein degradation by dBET1 ameliorates acute ischemic brain injury and improves functional outcomes associated with reduced neuroinflammation and oxidative stress and preservation of blood-brain barrier integrity.

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-1ß, 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.