Preprint: a zebrafish larva screen identified seven molecules that reversibly increase blood-brain barrier permeability
Preprint: a zebrafish larva screen identified seven molecules that reversibly increase blood-brain barrier permeability
On September 4, researchers at Rutgers University posted the second version of a preprint describing FishNAP, an assay conducted in zebrafish larvae. Among 2 320 FDA-approved small molecules, 11 candidates repeatedly altered the larvae’s behavior. Direct measurements then identified seven compounds that produced substantial leakage of a small marker into brain tissue. Three of these compounds were subsequently tested in adult mice.
The blood-brain barrier is formed by the cells lining blood vessels in the brain. These cells protect neural tissue and regulate exchange between the blood and the brain. When the barrier is intact, loperamide remains outside the brain, and the larvae retain their usual level of movement. A substance that temporarily increases the permeability of the vessel wall allows loperamide to enter the brain and slows the larvae’s movements. FishNAP uses this response as a rapid initial signal. The researchers then measure leakage of a fluorescent marker into brain tissue.
The researchers screened 2 320 molecules using this behavioral signal and retained 11 candidates that repeatedly produced the same effect. Eight significantly increased leakage of the small marker, and seven produced a substantial increase. All seven allowed the smallest marker used in the study to cross the barrier. Passage of the larger markers varied among compounds. The researchers selected the lowest dose that produced an effect. After each compound was removed, barrier function recovered within 24 hours for all seven compounds. These seven compounds passed both stages of testing, first the behavioral assay and then direct measurement of marker passage into the brain.
The researchers subsequently tested calcitriol, lovastatin, and sunitinib in adult mice. All three increased penetration of the small marker and accumulation of albumin, a blood protein, in brain tissue. Calcitriol and lovastatin also allowed immunoglobulin G, a larger protein, to cross the barrier. After treatment with each of the three compounds, the amounts of CLDN5 and MFSD2A decreased in the cerebral cortex. These proteins help vascular cells maintain the barrier. The amount of CAV1, which is associated with transport through cells in small vesicles, increased. The researchers interpret this combination as evidence of weaker tight junctions between vascular cells and increased transport through the cells.
In the BrainCAB study, a tag bound an antibody to CA-IV, a protein on the surface of brain blood vessels, and transported cargo from the blood into brain tissue in mice and newborn macaques. FishNAP identifies small molecules that temporarily alter vessel wall permeability. For this approach, the relevant factors are the size of the intended cargo and the specific property of the vessel wall altered by the identified compound.