Project description:The blood-brain barrier (BBB) is an evolutionary conserved tissue interface that possesses potent chemical protection properties functioning to strictly modulate the central nervous system (CNS) microenvironment. These same properties, including tight cellular junctions and efflux transporters, also limit access of CNS-active pharmaceuticals. For this reason, understanding the molecular mechanisms that regulate BBB chemical protection is of great biomedical interest. The BBB of Drosophila consists of two surface glia layers that completely surround the brain. This tissue interface contains both “tight” cellular junctions (termed septate junctions) and drug efflux transporters; thus, the Drosophila BBB can potentially serve as a model for understanding complex regulation of BBB physiology. In this study, we show reciprocal compensatory responses following disruption of critical BBB genes: deletion of the septate junction regulator Moody causes increased drug efflux and up-regulation of the P-glycoprotein ortholog Mdr65; conversely, disruption of Mdr65 expression causes increased septate junction tightness and up-regulation of Moody. We reveal these homeostatic interactions with physiologic observations, gene expression data, and anatomical images of the BBB surface. Whole brain microarray data point to responses that are consistent with our physiologic observations and these responses are likely localized to the BBB. To our knowledge, this is the first observation of a reciprocal compensatory interaction at a tissue barrier. Furthermore, this study paves the way for future studies elucidating the direct pathways that link GPCR signaling and drug transporter regulation at the BBB. The main comparisons are between WT_new, C17 (Moody null), and PMdr65 (Mdr65 null). Since WT_new were processed on a different day than C17 and PMdr65, we also included another WT sample (WT_old) to control for genes that change depending on batch effects. Since the mutants are also in a different genetic background than WT, we also included a control that is in a similar background (UAS_control).

Project description:The blood-brain barrier (BBB) dynamically controls and maintains a precisely balanced brain microenvironment necessary for reliable functioning. It is made up of highly specialized endothelial cells (ECs) in the lining of the vascular wall. These ECs are surrounded by the basal lamina, astrocytic perivascular endfeet, pericytes, microglia and neuronal processes that have been shown to contribute to barrier function1. In essence, the brain endothelium limits both transcellular and paracellular passage of cells and molecules into the central nervous system (CNS). Transcellular passage of hydrophilic molecules is limited due to a low rate of transcytotic vesicles, an extremely low pinocytotic activity, expression of active efflux transporters, and high metabolic activity. Paracellular diffusion of hydrophilic molecules and trafficking of immune cells is restricted by a network of tight junctions (TJs)2-5. Due to these characteristics, the BBB is able to protect the CNS from sudden changes in blood composition and uncontrolled influx of immune cells. In a large number of neuro-inflammatory diseases, an impaired function and an opening of the BBB are observed. In diseases of the CNS like multiple sclerosis or stroke or after brain trauma, the BBB becomes inflamed (mediated by for example reactive oxygen species (ROS) and hypoxia) and its function severely impaired which gives rise to enhanced cellular infiltration, thus contributing to tissue damage and neurological deficits. Due to the specialized nature of brain ECs, transmigration of leukocytes through cerebrovascular endothelium is likely to differ from that in other vascular beds. Generally, the transmigration process is closely controlled by the interaction of leukocytes with the endothelial surface followed by low velocity rolling, arrest, firm adhesion, and finally transmigration. For the diapedesis of immune cells across the cerebral vasculature a re-arrangement of the cytoskeleton and opening of the TJ complexes is required to allow cells to pass into the sub endothelial space. In the initial step of the adhesive cascade leukocytes adhere to the endothelium with low affinity. This adhesion is mediated by several members of the selectin family and their corresponding ligands. Despite the low affinity of these interactions, resulting contact of leukocytes with the endothelium leads to further activation of both cell types and finally transmigration of the leukocytes through the BBB6,7. The glycosylation of endothelial cells of different vascular bed origins To date it is unknown which specific carbohydrate structures on the BBB endothelium mediate leukocyte capture and rolling, and whether these structures are differentially expressed onto inflamed brain EC compared to normal brain ECs and vascular beds of other organs. Initial studies using qPCR and FACS analysis within our group reveal that brain endothelial cells have a different profile in their glycosylation-related genes compared to microvascular ECs upon inflammation, which may result in a different glycosylation profile of adhesive structures and may underlie rolling, adhesion and diapedesis of leukocytes. In this project we wish to identify specific, glycosylated structures on brain endothelial cells that mediate capture, rolling and diapedesis of leukocytes in the brain. To investigate the expression of glycosyltransferases in dendritic cells and the changes in expression associated with maturation. RNA preparations from stimulated and non-stimulated hcMEC/D3 (human brain endothelial cells line) and FMVEC (human promary microvascular endothelial cells isolated from foreskins) were sent to both Microarray Core (E) and Core(C). The RNA was put on an RNeasy Column, amplified, labeled, and hybridized to the Glycov3 microarrays. Data was sent to Dr. van Kooyk's lab for analysis.

Project description:The blood-brain barrier (BBB) dynamically controls and maintains a precisely balanced brain microenvironment necessary for reliable functioning. It is made up of highly specialized endothelial cells (ECs) in the lining of the vascular wall. These ECs are surrounded by the basal lamina, astrocytic perivascular endfeet, pericytes, microglia and neuronal processes that have been shown to contribute to barrier function1. In essence, the brain endothelium limits both transcellular and paracellular passage of cells and molecules into the central nervous system (CNS). Transcellular passage of hydrophilic molecules is limited due to a low rate of transcytotic vesicles, an extremely low pinocytotic activity, expression of active efflux transporters, and high metabolic activity. Paracellular diffusion of hydrophilic molecules and trafficking of immune cells is restricted by a network of tight junctions (TJs)2-5. Due to these characteristics, the BBB is able to protect the CNS from sudden changes in blood composition and uncontrolled influx of immune cells. In a large number of neuro-inflammatory diseases, an impaired function and an opening of the BBB are observed. In diseases of the CNS like multiple sclerosis or stroke or after brain trauma, the BBB becomes inflamed (mediated by for example reactive oxygen species (ROS) and hypoxia) and its function severely impaired which gives rise to enhanced cellular infiltration, thus contributing to tissue damage and neurological deficits. Due to the specialized nature of brain ECs, transmigration of leukocytes through cerebrovascular endothelium is likely to differ from that in other vascular beds. Generally, the transmigration process is closely controlled by the interaction of leukocytes with the endothelial surface followed by low velocity rolling, arrest, firm adhesion, and finally transmigration. For the diapedesis of immune cells across the cerebral vasculature a re-arrangement of the cytoskeleton and opening of the TJ complexes is required to allow cells to pass into the sub endothelial space. In the initial step of the adhesive cascade leukocytes adhere to the endothelium with low affinity. This adhesion is mediated by several members of the selectin family and their corresponding ligands. Despite the low affinity of these interactions, resulting contact of leukocytes with the endothelium leads to further activation of both cell types and finally transmigration of the leukocytes through the BBB6,7.

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Astroglial Connexin 43 sets the blood brain barrier immune quiescence

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