IL-1ß disrupts the initiation of blood-brain barrier development by inhibiting endothelial Wnt/ß-catenin signaling.

During neuroinflammation, the proinflammatory cytokine interleukin-1ß (IL-1ß) impacts blood-brain barrier (BBB) function by disrupting brain endothelial tight junctions, promoting vascular permeability, and increasing transmigration of immune cells. Here, we examined the effects of Il-1ß on the in vivo initiation of BBB development. We generated doxycycline-inducible transgenic zebrafish to secrete Il-1ß in the CNS. To validate the utility of our model, we showed Il-1ß dose-dependent mortality, recruitment of neutrophils, and expansion of microglia. Using live imaging, we discovered that Il-1ß causes a significant reduction in CNS angiogenesis and barriergenesis. To demonstrate specificity, we rescued the Il-1ß induced phenotypes by targeting the zebrafish il1r1 gene using CRISPR-Cas9. Mechanistically, we determined that Il-1ß disrupts the initiation of BBB development by decreasing Wnt/ß-catenin transcriptional activation in brain endothelial cells. Given that several neurodevelopmental disorders are associated with inflammation, our findings support further investigation into the connections between proinflammatory cytokines, neuroinflammation, and neurovascular development.

IL-1ß disrupts blood-brain barrier development by inhibiting endothelial Wnt/ß-catenin signaling.

During neuroinflammation, the proinflammatory cytokine Interleukin-1ß (IL-1ß) impacts blood-brain barrier (BBB) function by disrupting brain endothelial tight junctions, promoting vascular permeability, and increasing transmigration of immune cells. Here, we examined the effects of Il-1ß on the in vivo development of the BBB. We generated a doxycycline-inducible transgenic zebrafish model that drives secretion of Il-1ß in the CNS. To validate the utility of our model, we showed Il-1ß dose-dependent mortality, recruitment of neutrophils, and expansion of microglia. Using live imaging, we discovered that Il-1ß causes a significant reduction in CNS angiogenesis and barriergenesis. To demonstrate specificity, we rescued the Il-1ß induced phenotypes by targeting the zebrafish il1r1 gene using CRISPR/Cas9. Mechanistically, we determined that Il-1ß disrupts BBB development by decreasing Wnt/ß-catenin transcriptional activation in brain endothelial cells. Given that several neurodevelopmental disorders are associated with inflammation, our findings support further investigation into the connections between proinflammatory cytokines, neuroinflammation, and neurovascular development.

Brain endothelial cells acquire blood-brain barrier properties in the absence of Vegf-dependent CNS angiogenesis.

During neurovascular development, brain endothelial cells (BECs) respond to secreted signals from the neuroectoderm that regulate CNS angiogenesis, the formation of new blood vessels in the brain, and barriergenesis, the acquisition of blood-brain barrier (BBB) properties. Wnt/ß-catenin signaling and Vegf signaling are both required for CNS angiogenesis; however, the relationship between these pathways is not understood. Furthermore, while Wnt/ß-catenin signaling is essential for barriergenesis, the role of Vegf signaling in this vital process remains unknown. Here, we provide the first direct evidence, to our knowledge, that Vegf signaling is not required for barriergenesis and that activation of Wnt/ß-catenin in BECs is independent of Vegf signaling during neurovascular development. Using double transgenic glut1b:mCherry and plvap:EGFP zebrafish (Danio rerio) to visualize the developing brain vasculature, we performed a forward genetic screen and identified a new mutant allele of kdrl, an ortholog of mammalian Vegfr2. The kdrl mutant lacks CNS angiogenesis but, unlike the Wnt/ß-catenin pathway mutant gpr124, acquires BBB properties in BECs. To examine Wnt/ß-catenin pathway activation in BECs, we chemically inhibited Vegf signaling and found robust expression of the Wnt/ß-catenin transcriptional reporter line 7xtcf-Xla.Siam:EGFP. Taken together, our results establish that Vegf signaling is essential for CNS angiogenesis but is not required for Wnt/ß-catenin-dependent barriergenesis. Given the clinical significance of either inhibiting pathological angiogenesis or stimulating neovascularization, our study provides valuable new insights that are critical for the development of effective therapies that target the vasculature in neurological disorders.

Functional identification of the zebrafish Interleukin-1 receptor in an embryonic model of Il-1ß-induced systemic inflammation.

Interleukin-1ß (IL-1ß) is a potent proinflammatory cytokine that plays a vital role in the innate immune system. To observe the innate immune response in vivo, several transgenic zebrafish lines have been developed to model IL-1ß-induced inflammation and to visualize immune cell migration and proliferation in real time. However, our understanding of the IL-1ß response in zebrafish is limited due to an incomplete genome annotation and a lack of functional data for the cytokine receptors involved in the inflammatory process. Here, we use a combination of database mining, genetic analyses, and functional assays to identify zebrafish Interleukin-1 receptor, type 1 (Il1r1). We identified putative zebrafish il1r1 candidate genes that encode proteins with predicted structures similar to human IL1R1. To examine functionality of these candidates, we designed highly effective morpholinos to disrupt gene expression in a zebrafish model of embryonic Il-1ß-induced systemic inflammation. In this double transgenic model, ubb:Gal4-EcR, uas:il1ßmat , the zebrafish ubiquitin b (ubb) promoter drives expression of the modified Gal4 transcription factor fused to the ecdysone receptor (EcR), which in turn drives the tightly-regulated expression and secretion of mature Il-1ß only in the presence of the ecdysone analog tebufenozide (Teb). Application of Teb to ubb:Gal4-EcR, uas:il1ßmat embryos causes premature death, fin degradation, substantial neutrophil expansion, and generation of reactive oxygen species (ROS). To rescue these deleterious phenotypes, we injected ubb:Gal4-EcR, uas:il1ßmat embryos with putative il1r1 morpholinos and found that knockdown of only one candidate gene prevented the adverse effects caused by Il-1ß. Mosaic knockout of il1r1 using the CRISPR/Cas9 system phenocopied these results. Taken together, our study identifies the functional zebrafish Il1r1 utilizing a genetic model of Il-1ß-induced inflammation and provides valuable new insights to study inflammatory conditions specifically driven by Il-1ß or related to Il1r1 function in zebrafish.

Evoked potentials as a biomarker of remyelination.

Multiple sclerosis (MS) is a common cause of neurologic disease in young adults that is primarily treated with disease-modifying therapies which target the immune and inflammatory responses. Promotion of remyelination has opened a new therapeutic avenue, but how best to determine efficacy of remyelinating drugs remains unresolved. Although prolongation and then shortening of visual evoked potential (VEP) latencies in optic neuritis in MS may identify demyelination and remyelination, this has not been directly confirmed. We recorded VEPs in a model in which there is complete demyelination of the optic nerve, with subsequent remyelination. We examined the optic nerves microscopically during active disease and recovery, and quantitated both demyelination and remyelination along the length of the nerves. Latencies of the main positive component of the control VEP demonstrated around 2-fold prolongation during active disease. VEP waveforms were nonrecordable in a few subjects or exhibited a broadened profile which precluded peak identification. As animals recovered neurologically, the VEP latencies decreased in association with complete remyelination of the optic nerve but remained prolonged relative to controls. Thus, it has been directly confirmed that VEP latencies reflect the myelin status of the optic nerve and will provide a surrogate marker in future remyelination clinical trials.