This Is MS Multiple Sclerosis Community: Knowledge & Support

Dr. Frédéric Charron, researcher at the Institut de recherches cliniques de Montréal (IRCM), and his team have shown for the first time that a key molecule of the vascular system directs axons during the formation of neural circuits. This connection between the nervous system and the vascular system could be a good starting point for the development of therapies for neurodegenerative diseases. The discovery will be published June 9 by Neuron.

Highlights
VEGF is secreted by the floor plate
Haplodeficiency of Vegf in the floor plate causes axon guidance defects in vivo
Inactivation of Flk1 in commissural neurons causes axon guidance defects in vivo
VEGF/Flk1 activates Src family kinases and induces commissural axon turning in vitro
Summary

Growing axons are guided to their targets by attractive and repulsive cues. In the developing spinal cord, Netrin-1 and Shh guide commissural axons toward the midline. However, the combined inhibition of their activity in commissural axon turning assays does not completely abrogate turning toward floor plate tissue, suggesting that additional guidance cues are present. Here we show that the prototypic angiogenic factor VEGF is secreted by the floor plate and is a chemoattractant for commissural axons in vitro and in vivo. Inactivation of Vegf in the floor plate or of its receptor Flk1 in commissural neurons causes axon guidance defects, whereas Flk1 blockade inhibits turning of axons to VEGF in vitro. Similar to Shh and Netrin-1, VEGF-mediated commissural axon guidance requires the activity of Src family kinases. Our results identify VEGF and Flk1 as a novel ligand/receptor pair controlling commissural axon guidance.

Abstract

Flk-1 (human counterpart, KDR) tyrosine kinase, which is one of the two VEGF receptors, is crucial for vascular development. Recently, we showed that, among tyrosine residues of KDR, tyrosine residues 1175 (Y1175, corresponding to Y1173 in murine Flk-1) and Y1214 (Y1212 in Flk-1) are autophosphorylated in response to VEGF, and that Y1175 is important for VEGF-dependent phospholipase Cγ/PKC/mitogen-activated protein kinase activation leading to DNA synthesis in cultured endothelial cells. However, the importance of these tyrosine residues in Flk-1/KDR in vivo is not yet known. To examine the role of these Flk-1 tyrosine residues in vivo, we generated knock-in mice substituting Y1173 and Y1212 of the Flk-1 gene with phenylalanine, respectively. As a result, Flk-11173F homozygous mice died between embryonic days 8.5 and 9.5 without any organized blood vessels or yolk sac blood islands, and hematopoietic progenitors were severely reduced, similar to the case of Flk-1 null mice. In contrast, Flk-11212F homozygous mice were viable and fertile. These results suggest that the signaling via Y1173 of Flk-1 is essential for endothelial and hematopoietic development during embryogenesis.

DISCUSSION
The primary finding of this investigation is that impairment of Flk-1-mediated PI3-kinase/Akt signaling contributes to the age-induced reduction of flow-dependent, NO-mediated vasodilation in coronary arterioles from male Fischer-344 rats. This finding confirms earlier observations of reduced NO-mediated vasodilation in coronary resistance arteries of middle-aged rats (6) and provides novel insight into the effects of aging on cellular signaling mechanisms that activate eNOS in coronary arterioles. Our current findings indicate that age-related decrements in several components of the Flk-1 PI3-kinase/Akt signaling pathway contribute to the loss of flow- and VEGF-induced vasodilation that occurs with age in coronary arterioles. In contrast, ACh-induced signaling through G protein-coupled receptors is maintained with age, suggesting that Flk-1-mediated signaling is a critical target in age-induced coronary endothelial dysfunction.

Shear stress is a potent physiological stimulus for release of NO and is dependent on an intact endothelium (22). Flow-induced vasodilation is a key mediator of local vascular control in the coronary circulation (34, 35) and is critically dependent on endothelium-dependent release of NO, as evidenced by reports demonstrating that nitric oxide synthase inhibition eliminates flow-induced vasodilation in coronary arterioles of several species (25, 30, 41). Recent studies have indicated that VEGFR2, also known as Flk-1 (4), is rapidly tyrosine phosphorylated by flow (18, 24) and VEGF (14), leading to PI3-kinase-Akt-eNOS activation. Our results show that flow- and VEGF-induced vasodilatory responses of coronary arterioles are dramatically reduced by inhibition of Flk-1 with SU-1498. Importantly, our current results show that exposure to flow increases phosphorylation of Flk-1 in coronary arterioles from young rats, but not in coronary arterioles from old rats. Thus our results indicate that flow-induced vasodilation is impaired in coronary arterioles of senescent rats (24 mo) and extend previous findings in vessels from cardiac (6) and skeletal (29, 42) muscle by demonstrating that deficits in Flk-1/PI3-kinase/Akt signaling contribute to age-related reductions of NO-mediated vasodilation in coronary arterioles. Our current work also indicates that the decrease in Flk-1 activation that occurs with age is accompanied by a decrease in Flk-1 protein levels.

"This research could have an important long-term impact in the field of spinal cord repair, as the results will help us better understand the development of the spinal cord," says Dr. Charron, Director of the IRCM's Molecular Biology of Neural Development research unit. "The more we learn about the molecules needed to appropriately guide axons, the more it will become possible to develop a therapy to treat spinal cord injuries."