Hypoxia Disruption of Vertebrate CNS Pathfinding through EphrinB2 Is Rescued by Magnesium

Abstract

<div><p>The mechanisms of hypoxic injury to the developing human brain are poorly understood, despite being a major cause of chronic neurodevelopmental impairments. Recent work in the invertebrate <em>Caenorhabditis elegans</em> has shown that hypoxia causes discrete axon pathfinding errors in certain interneurons and motorneurons. However, it is unknown whether developmental hypoxia would have similar effects in a vertebrate nervous system. We have found that developmental hypoxic injury disrupts pathfinding of forebrain neurons in zebrafish (<em>Danio rerio</em>), leading to errors in which commissural axons fail to cross the midline. The pathfinding defects result from activation of the hypoxia-inducible transcription factor (<em>hif1</em>) pathway and are mimicked by chemical inducers of the <em>hif1</em> pathway or by expression of constitutively active <em>hif1α</em>. Further, we found that blocking transcriptional activation by <em>hif1α</em> helped prevent the guidance defects. We identified <em>ephrinB2a</em> as a target of <em>hif1</em> pathway activation, showed that knock-down of <em>ephrinB2a</em> rescued the guidance errors, and showed that the receptor <em>ephA4a</em> is expressed in a pattern complementary to the misrouting axons. By targeting a constitutively active form of <em>ephrinB2a</em> to specific neurons, we found that <em>ephrinB2a</em> mediates the pathfinding errors via a reverse-signaling mechanism. Finally, magnesium sulfate, used to improve neurodevelopmental outcomes in preterm births, protects against pathfinding errors by preventing upregulation of <em>ephrinB2a</em>. These results demonstrate that evolutionarily conserved genetic pathways regulate connectivity changes in the CNS in response to hypoxia, and they support a potential neuroprotective role for magnesium.</p> </div