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A limiting feature of many neurological therapies is the ability of molecules to cross the blood-brain barrier (BBB) from the circulatory system. Since the BBB prevents simple diffusion of materials across the divide, identifying the proteins responsible for transport is necessary for effective design of BBB-crossing therapies.

“So basically, everything in the circulating blood, if they want to have an exchange with the organ, they need to pass through this interface,” says senior author Jiefu Li, PhD, Janelia Research Campus Group Leader at the Howard Hughes Medical Institute.

Identification of the structures within blood vessels involved with the processes of molecular movement across the BBB has been somewhat elusive. However, Li and his team have developed a technique that not only identifies proteins within the luminal surface—the inner lining—of the vasculature, but also works in vivo, allowing them to track how these features change across the aging brain.

“Understanding how the blood-brain barrier works, particularly figuring out the molecular targets that you can play with to open and close the barrier, will provide new possibilities for drug delivery,” Li says.

Their work is published in Science in a paper entitled, “Luminal surface proteome of the brain vasculature uncovers blood-brain barrier regulators.”

Using mice, the team developed a proteomic profiling method that can be used not only in brain vasculature, but throughout the body. “Briefly, a lectin-conjugated peroxidase is perfused and anchored to the luminal surface of blood vessels to catalyze the biotinylation of adjacent proteins, thereby enabling subsequent protein enrichment and mass spectrometry analysis,” wrote the authors.

They tested the method in the brain, kidney and intestine, in both mice and northern tree shrew, showing functionality and applicability across organs and species.

“This will allow us to say: we know that the vasculature system is doing different things in different organs and it relies on this luminal surface, but how does that happen? What are the molecular players there?” Li says.

Using quantitative proteomics of the luminal surface—from early development through adulthood and aging—they found that over time there was a decrease in angiogenic and transport proteins. They also found an increase in proteins that increased stiffness in the vasculature.

In addition to developing this in vivo technique, the team identified two proteins that are temporally distinct in their expression while both playing a role in modulating BBB permeability. Knockouts of nitric oxide synthase Nos3 and arginine transporter Slc7a1 resulted in BBB leakage in neonates, but not adults, while genetic screens identified hyaluronidase HYAL2 as being required for maintaining BBB integrity throughout the lifespan of mice.

“What we know now is that we have two new pathways, potentially, to open the blood-brain barrier and to inform some therapeutic developments,” says Li.

Utilization of this proteomic based method in vivo both opens up new avenues of functional research across the cardiovascular system, and also provides data and a methodology for novel therapeutic targets for crossing the BBB.

“This method solves an important need but it’s also a very easy-to-use method, so everyone can use it,” Li says.

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