Microvascular Abnormalities in Adrenoleukodystrophy: Effects of ABCD1 Deficiency Upon Brain Endothelium. (S35.005)

OBJECTIVE:We investigated the impact of ABCD1 deficiency upon BBB permeability in primary human brain microvascular endothelial cells (HBMECs) and autopsy samples from patients with Cerebral X-linked adrenoleukodystrophy. BACKGROUND:Cerebral X-linked adrenoleukodystrophy (CALD) is a neurodegenerative disorder resulting from mutations in the ABCD1 gene. The disease manifests as progressive inflammatory demyelination suggesting that blood brain barrier (BBB) disruption plays an important role in lesion progression. Normal BBB permeability depends on the integrity of tight junction proteins and regulation of adhesion molecules.
 METHODS: We performed immunofluorescence of autopsy brain samples and quantified the protein expression in different zones of the lesion (cortex, perilesional, and core) using ImageJ software. Endothelial cells were treated with ABCD1 siRNA and changes in mRNA and protein expression were assessed by QPCR and western blot, respectively. HBMECs and THP1 cells were used in adhesion and flow adhesion assays, to determine functional implications of ABCD1 silencing.
 RESULTS:In CALD specimens, we found abnormal expression of ZO-1 and Claudin-5 in endothelial cells with redistribution to microglia and macrophages at the inflammatory edge and core of the lesion. ABCD1 silencing led to increased ICAM1 expression, further upregulated following TNF-alpha stimulation. It also led to a significant decrease of Claudin-5 and ZO-1. Immunofluorescence confirmed the displacement of Claudin-5 from the tight junctions of the cell membrane. Interestingly, these effects of ABCD1 silencing were not present in Human Umbilical Vascular Endothelial Cells (HUVECs), indicating a specific vulnerability of brain microvascular endothelial cells. Consequently, we found increased adhesion of leukocytes following ABCD1 silencing in HBMECs. CONCLUSIONS:These results suggest a crucial role of ABCD1 dysfunction in brain endothelium, where functional changes may mediate abnormal migration of leukocytes to the brain and lead to subsequent demyelination. Our findings thus have the potential to identify new therapeutic targets for this devastating neurologic disorder. Study Supported by:NIH K08NS52550, R25NS065743, K12NS066225. Disclosure: Dr. Musolino has nothing to disclose. Dr. Gong has nothing to disclose. Dr. Juliet has nothing to disclose. Dr. Jimenez has nothing to disclose. Dr. Grabowski has nothing to disclose. Dr. Lok has nothing to disclose. Dr. Eng H has nothing to disclose. Dr. Frosch has received royalty payments from Elsevier. Dr. Eichler has received personal compensation for activities with Retrophin, Inc.