Cross species molecular analysis of glial and endothelial blood-brain barriers - PROJECT SUMMARY The blood-brain barrier (BBB) is characterized by a series of properties that tightly regulate the transport of ions, molecules and cells between the blood vessel lumen and the brain parenchyma. This BBB is critical to control the environment of the brain to allow for proper neuronal function, and to protect the brain from injury and disease. Dysregulation of the BBB has been implicated in a variety of disorders including stroke, Alzheimer’s disease, multiple sclerosis, and epilepsy. In addition, the BBB is a key obstacle to central nervous system (CNS) drug delivery. The BBB has been extensively studied in a small number of model organisms (mouse, zebrafish) in which endothelial cells form this barrier. Interestingly, several species have a BBB formed instead by glial cells including invertebrates, elasmobranchs (sharks, rays and relatives) and sturgeons, but very little is known about the molecular mechanisms of BBB function in these species. Furthermore, a lack of correspondence between phylogenetic relationships and endothelial versus glial BBB suggests that the BBB evolved independently several times. Hypotheses that may explain these observations include an ancestral vertebrate with a glial BBB and independent evolution of the endothelial BBB at least six times (hypothesis A), an ancestral vertebrate with an endothelial BBB and independent re-evolution of the glial BBB at least twice (hypothesis B), or an ancestral vertebrate in which endothelial and glial cells each accomplished aspects of BBB function, with current BBB diversity a result of a “push-and-pull” toward either extreme (hypothesis C). Detailed molecular knowledge of the BBB in non-model organisms is required to better understand the mechanisms underlying BBB function in vertebrates and their evolution. To achieve this we will employ single nucleus RNA-sequencing (snRNA-seq) of brain tissue in fish species with an endothelial BBB (hagfish, trout, zebrafish, and lungfish) and fish with a glial BBB (shark, ray, and sturgeon). We will also profile mouse and octopus as outgroup comparators. This will allow us to define the molecular characteristics of endothelial and glial cells in each species and perform a cross-species comparison of BBB-forming cell types. Furthermore, we will examine BBB function and ultrastructure by injecting horseradish peroxidase (HRP) systemically into each species and visualizing HRP localization with electron microscopy. Together, the proposed study will provide unprecedented new information related to the molecular foundations of BBB function across life and will yield new information on the evolution of the BBB. The resulting information may also reveal novel molecular targets for CNS drug delivery and for treating BBB dysfunction in CNS disorders.
