New methods to study blood-brain barrier permeability

Abstract

The blood-brain barrier (BBB) is essential for brain health, yet the biological mechanisms that impart and maintain BBB integrity remain incompletely understood. It has long been appreciated that BBB function is compromised in diverse diseases of the central nervous system (CNS), and new evidence that BBB dysfunction plays a causative role in disease pathology is emerging. Yet, a significant constraint on research into BBB biology is the lack of sensitive, reliable tools to measure BBB function. For my dissertation, I developed two new methods to measure BBB permeability in mouse models. The first method is an alternative to the classic BBB tracer assay in which leakage of an intravenous dye into the brain indicates pathological BBB permeability. My new method, HaloTrace, leverages the HaloTag ligand-receptor tool to generate a precise spatiotemporal readout of BBB integrity that avoids major pitfalls of existing methods. I present evidence that the fluorescent HaloTag ligand has minimal interactions with blood contents but can enter the brain specifically at sites of BBB dysfunction, where it covalently binds to nearby HaloTag receptors. The ligand accumulates in the brain during its short lifetime in circulation and is stably anchored in place for at least 24 hours. Free ligand is not retained in the blood vessels at detectable levels, so the entirety of ligand fluorescence represents true BBB leakage. Furthermore, I demonstrate that HaloTrace can quantify BBB permeability at multiple discrete timepoints prior to the experiment endpoint, offering flexibility in experimental design that current methods do not. This flexibility will be particularly useful for characterizing the spatiotemporal dynamics of leakage in longitudinal mouse studies. I also endeavored to measure the transport of serum proteins across the BBB. While fluorescent tracers are useful for detecting gross BBB breakdown, it is also advantageous to measure more nuanced aspects of BBB function like the active transport of endogenous proteins from the blood to the brain. Uncovering the identities of proteins shuttling across the BBB could lead to new CNS therapeutic delivery strategies or diagnose specific defects in BBB function in CNS disease models. With these goals in mind, I tested two methods to quantify the trafficking of endogenous proteins across the BBB. I successfully implemented two strategies to label endogenous serum proteins derived from liver hepatocytes: cell-type specific non-canonical amino acid tagging and proximity biotinylation. I show that proximity-biotinylation of secreted proteins with TurboID shows promise but will require further optimization before it is a sufficiently sensitive readout of protein transport at the BBB.