Frataxin-deficient human brain microvascular endothelial cells lose polymerized actin and are paracellularly permeable –implications for blood-brain barrier integrity in Friedreich’s Ataxia

BACKGROUND: Friedreich’s Ataxia (FRDA) is the most prevalent inherited ataxia; the disease results from loss of Frataxin, an essential mitochondrial iron trafficking protein. FRDA presents as neurodegeneration of the dorsal root ganglion and cerebellar dentate nuclei, followed by brain iron accumulation in the latter. End stage disease includes cardiac fibrosis that contributes to hypertrophic cardiomyopathy. The microvasculature plays an essential barrier role in both the brain and heart, thus an investigation of this tissue system in FRDA is essential to the delineation of the cellular dysfunction in this genetic disorder. Here, we investigate brain microvascular endothelial cell integrity in FRDA in a model of the blood-brain barrier (BBB). METHODS: We used lentiviral mediated shRNA delivery to generate a novel FRDA model in immortalized human brain microvascular endothelial cells (hBMVEC) that compose the microcapillaries of the BBB. We verified known cellular pathophysiologies of FXN knockdown including increased oxidative stress, loss of energy metabolism, and increased cell size. Furthermore, we investigated cytoskeletal architecture including the abundance and organization of filamentous actin, and barrier physiology via transendothelial electrical resistance and fluorescent tracer flux. RESULTS: shFXN hBMVEC display the known FRDA cell morbidity including increased oxidative stress, decreased energy metabolism, and an increase in cell size. We demonstrate that shFXN hBMVEC have less overall filamentous actin, and that filamentous actin is lost at the cell membrane and cortical actin ring. Consistent with loss of cytoskeletal structure and anchorage, we found decreased barrier strength and increased paracellular tracer flux in the shFXN hBMVEC transwell model. CONCLUSION: We identified that insufficient FXN levels in the hBMVEC BBB model causes changes in cytoskeletal architecture and increased barrier permeability, cell pathologies that may be related to patient brain iron accumulation, neuroinflammation, neurodegeneration, and stroke. Our findings implicate other barrier cells, e.g., the cardiac microvasculature, likely contributory also to disease pathology in FRDA.