Dritan Agalliu

Molecular, cellular and genetic analysis of mammalian blood-brain barrier development and the role of the barrier in disease pathogenesis – My laboratory is investigating two fundamental issues in the biology of the mammalian blood-brain barrier (BBB): 1) the mechanisms governing development and maintenance of the barrier; and 2) how structural components of the BBB are affected in diseases of the brain and spinal cord where barrier function is impaired.

Endothelial cells that line the blood vessels of the mammalian central nervous system (CNS) form a unique, tight barrier that maintains the homeostasis necessary for neuronal and glial function, and protects the CNS from pathological agents and immune cell invasion. Barrier properties of CNS endothelial cells (ECs) are mediated by three distinct cell biological mechanisms: a) extremely tight junctions that prevent diffusion of small molecules between endothelial cells (paracellular pathway); b) very few endocytotic vesicles (caveolae) that transcytose slowly and thereby reduce transport of large molecules across the brain endothelium (transcellular pathway); and c) transporters that shuttle only selected molecules between the blood and the brain (Figure 1). This barrier breaks down during CNS insults such as trauma and stroke, or in neurodegenerative disorders such as multiple sclerosis (MS).

What are the cellular and molecular mechanisms that govern development and maintenance of the BBB?
We are interested in identifying key signaling pathways that regulate formation of the BBB, because the molecular mechanisms that control this process are poorly understood and have obvious implications for repairing BBB function for CNS diseases in which the barrier breaks down. We have found that the Wnt/-catenin pathway plays an important role in regulating both CNS angiogenesis and some properties of the barrier. We have identified a novel inhibitor of this pathway named Apcdd1 that is expressed in CNS endothelial cells when they acquire their barrier properties during development. We are currently investigating the roles of Apcdd1 in CNS angiogenesis and blood-brain barrier development, using mouse strains that lack Apcdd1 function and transgenic mice that overexpress Apcdd1 in endothelial cells. Our group is also interested in identifying novel signaling molecules that are secreted by CNS progenitors, pericytes and astrocytes that contribute to formation and maintenance of the barrier. We have developed methods to culture endothelial cells from the CNS with other cell types, in order to identify which cells and signaling pathways induce distinct functional components of the BBB, such as tight junctions and low rates of transcytosis. Finally, we are also interested in understanding how tight junctions between endothelial cells in the CNS develop and mature during BBB formation. We have developed proteomic discovery platforms for: a) identifying novel junctional proteins important for barrier development and function; and b) potential BBB structural modifications that underlie CNS diseases exhibiting reduced barrier function.

How are structural components of the BBB affected in diseases of the brain and spinal cord, where barrier function is impaired?
Remarkably little is known about how the BBB breaks down in CNS diseases that have various pathological origins such as hypoxia and stroke, or in neurodegenerative diseases such as multiple sclerosis. In particular, the roles of BBB structural components such as tight junctions and transcytosis vesicles (caveolae) that allow small or large molecules, respectively, to cross the BBB are poorly defined for the pathogenesis of brain disorders. We have created two novel transgenic reagents: a) a strain where tight junctions are labeled with GFP (Tie2p::eGFP::Claudin-5); and b) a strain where caveolae can be visualized with the red fluorescent protein pmKate2 (Tie2p::pmKate2::Caveolin-1). For the first time, this allows real-time analysis of tight junction dynamics and changes in EC transcytosis rates for a variety of CNS disease models where the BBB is compromised. We are using these novel transgenic mice to image tight junctions in brain capillaries using two-photon microscopy in vivo, combined with quantitative analysis of fluorescent tracer leakage across the BBB (Figure 2). We have initiated these studies in the mouse model for stroke, and have found that although BBB function is impaired as early as 6 hours post-stroke, tight junctions are very stable in the stroke core region during the first 24 hours after the insult. These findings indicate that transcytosis may be the major pathway that impairs BBB function during stroke. We are currently extending these approaches to analyze Experimental Autoimmune Encephalitis (EAE), a mouse model for multiple sclerosis (MS).

We are also conducting proteomic analyses of tight junctions for various murine disease model (stroke or EAE) that displays impaired barrier function. This will identify how a key structural component of the BBB, namely the tight junction, is regulated during CNS diseases that exhibit BBB breakdown, as well as elucidate similarities and differences in BBB function that are present in two very different diseases.

How are cellular interactions within the neurovascular unit impaired during neurodegenerative disease pathogenesis?
My laboratory performs two-photon imaging using transgenic mouse lines in which various components of the neurovascular unit are differentially labeled with fluorescent proteins, in order to examine dynamic changes in cellular interactions during the progression of CNS diseases such as stroke or EAE. Initially, we are focusing on interactions of pericytes with endothelial cells in vivo during stroke or EAE progression, in order to test the hypothesis that detachment of pericytes from CNS ECs is an early event that promotes CNS pathologies, expecially for MS. We employ doubly transgenic mice containing both the eGFP::Claudin-5 and CSPG::dsRed2 BAC markers, which label EC junctions in green and pericytes in red. Similar experiments are being carried out with Tie2p::eGFP::Caveolin-1 mice to examine if pericyte detachment correlates with an increased rate of EC transcytosis. We will ultimately extend these studies to investigate interactions of ECs with microglia, astrocytes and immune cells during stroke or EAE progression.

Recent Publications