Na+/H+ Exchanger 9 Regulates Iron Mobilization at the Blood-Brain Barrier in Response to Iron Starvation

Abstract

Edited by Paul E. FraserIron is essential for brain function, with loss of iron homeostasis in the brain linked to neurological diseases ranging from rare syndromes to more common disorders, such as Parkinson's and Alzheimer's diseases. Iron entry into the brain is regulated by the blood-brain barrier (BBB). Molecular mechanisms regulating this transport are poorly understood. Using an in vitro model of the BBB, we identify NHE9, an endosomal cation/proton exchanger, as a novel regulator of this system. Human brain microvascular endothelial cells (hBMVECs) that constitute the BBB receive brain iron status information via paracrine signals from ensheathing astrocytes. In hBMVECs, we show that NHE9 expression is up-regulated very early in a physiological response invoked by paracrine signals from iron-starved astrocytes. Ectopic expression of NHE9 in hBMVECs without external cues induced up-regulation of the transferrin receptor (TfR) and down-regulation of ferritin, leading to an increase in iron uptake. Mechanistically, we demonstrate that NHE9 localizes to recycling endosomes in hBMVECs where it raises the endosomal pH. The ensuing alkalization of the endosomal lumen increased translocation of TfRs to the hBMVEC membrane. TfRs on the membrane were previously shown to facilitate both recycling-dependent and -independent iron uptake. We propose that NHE9 regulates TfR-dependent, recycling-independent iron uptake in hBMVECs by fine-tuning the endosomal pH in response to paracrine signals and is therefore an important regulator in iron mobilization pathway at the BBB.There is high demand for iron in the brain, one of the most metabolically active organs in the body (1, 2). Iron is a co-factor of several proteins involved in specialized brain cell functions such as synthesis of neurotransmitters and myelination (3-5). Iron is also necessary for housekeeping functions such as mitochondrial respiration and DNA synthesis, crucial for brain function (5). Insufficient or surplus iron in the brain has been associated with various neurological diseases (6 -10). Although iron deficiency is associated with cognitive and brain structural deficits, excess brain iron is implicated in Alzheimer's, Parkinson's, and other neurodegenerative diseases (10, 11). Therefore, it is not surprising that iron levels are tightly regulated in the brain (10).Iron entry into the brain is regulated by the blood-brain barrier (BBB) 3 (10). This physiological barrier is characterized by tight junctions between brain microvascular endothelial cells (BMVECs) that block passive diffusion of iron into the brain (12). BMVECs are polarized cells with one surface (apical) facing the blood and the other (basal or abluminal) facing interstitial fluid of the brain. BMVECs and ensheathing astrocytes form the regulatory axis of the BBB, modulating iron transport from blood into the brain interstitium (2, 13). It is now known that BMVECs are the focal point for regulation of cerebral iron homeostasis and not mere conduits for iron transport (14). Paracrine fac...

A gain of function paradox: Targeted therapy for glioblastoma associated with abnormal NHE9 expression

“…Cells were mounted onto slides using Prolong gold antifade reagent (Invitrogen). Immunostaining of human brain microvascular endothelial cells (BMVECs) in culture with RAW264.7 cells was conducted as described previously . Anti‐human von Willebrand factor antibody (DakoCytomation) was used as a marker for BMVECs.…”

“…The cells were then fixed for 20 minutes at room temperature with solution containing 4% paraformaldehyde and 4% sucrose in PBS, following previously published protocol. 10 Cells were mounted onto slides using Prolong gold antifade reagent (Invitrogen). Immunostaining of human brain microvascular endothelial cells (BMVECs) in culture with RAW264.7 cells was conducted as described previously.…”

“…Immunostaining of human brain microvascular endothelial cells (BMVECs) in culture with RAW264.7 cells was conducted as described previously. 10 Anti-human von Willebrand factor antibody (DakoCytomation) was used as a marker for BMVECs. All slides were imaged using Lumascope-620 microscope (Etaluma).…”

Nanotherapy for Alzheimer's disease and vascular dementia: Targeting senile endothelium

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