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The vascular endothelial growth factor (VEGFA, VEGF) regulates neurovascular patterning. Alternative splicing of the Vegfa gene gives rise to three major isoforms termed VEGF121, VEGF165 and VEGF189. VEGF165 binds the transmembrane protein neuropilin 1 (NRP1) and promotes the migration, survival and axon guidance of subsets of neurons, whereas VEGF121 cannot activate NRP1-dependent neuronal responses. By contrast, the role of VEGF189 in NRP1-mediated signalling pathways has not yet been examined. Here, we have combined expression studies and in situ ligand-binding assays with the analysis of genetically altered mice and in vitro models to demonstrate that VEGF189 can bind NRP1 and promote NRP1-dependent neuronal responses.

The semaphorins are the largest family of repulsive axon guidance molecules. Secreted semaphorins bind neuropilin receptors and repel sensory, sympathetic and motor axons. Here we show that CA1, CA3 and dentate gyrus axons from E15-E17 mouse embryo explants are selectively repelled by entorhinal cortex and neocortex. The secreted semaphorins Sema III and Sema IV and their receptors Neuropilin-1 and -2 are expressed in the hippocampal formation during appropriate stages. Sema III and Sema IV strongly repel CA1, CA3 and dentate gyrus axons; entorhinal axons are only repelled by Sema III. An antibody against Neuropilin-1 blocks the repulsive action of Sema III and the entorhinal cortex, but has no effect on Sema IV-induced repulsion. Thus, chemorepulsion plays a role in axon guidance in the hippocampus, secreted semaphorins are likely to be responsible for this action, and the same axons can be repelled by two distinct semaphorins via two different receptors.

Neuropilins (NRPs) are single-pass transmembrane receptors involved in several signaling pathways that regulate key physiological processes such as vascular morphogenesis and axon guidance. The MAM domain of NRP, which has previously been implicated in receptor multimerization, was the only portion of the ectopic domain of the NRPs for which the structure, until now, has been elusive. Using site-directed mutagenesis in the linker region preceding the MAM domain we generated a protein construct amenable to crystallization. Here we present the crystal structure of the MAM domain of human NRP1 at 2.24 A resolution. The protein exhibits a jellyroll topology, with Ca2+ ions bound at the inter-strand space enhancing the thermostability of the domain. We show that the MAM domain of NRP1 is monomeric in solution and insufficient to drive receptor dimerization, which leads us to propose a different role for this domain in the context of NRP membrane assembly and signaling.

Background After a spinal cord lesion, axon regeneration is inhibited by the presence of a diversity of inhibitory molecules in the lesion environment. At and around the lesion site myelin-associated inhibitors, chondroitin sulfate proteoglycans (CSPGs) and several axon guidance molecules, including all members of the secreted (class 3) Semaphorins, are expressed. Interfering with multiple inhibitory signals could potentially enhance the previously reported beneficial effects of blocking single molecules. RNA interference (RNAi) is a tool that can be used to simultaneously silence expression of multiple genes. In this study we aimed to employ adeno-associated virus (AAV) mediated expression of short hairpin RNAs (shRNAs) to target all Semaphorin class 3 signaling by knocking down its receptors, Neuropilin 1 (Npn-1) and Neuropilin 2 (Npn-2). Results We have successfully generated shRNAs that knock down Npn-1 and Npn-2 in a neuronal cell line. We detected substantial knockdown of Npn-2 mRNA when AAV5 viral vector particles expressing Npn-2 specific shRNAs were injected in dorsal root ganglia (DRG) of the rat. Unexpectedly however, AAV1-mediated expression of Npn-2 shRNAs and a control shRNA in the red nucleus resulted in an adverse tissue response and neuronal degeneration. The observed toxicity was dose dependent and was not seen with control GFP expressing AAV vectors, implicating the shRNAs as the causative toxic agents. Conclusions RNAi is a powerful tool to knock down Semaphorin receptor expression in neuronal cells in vitro and in vivo. However, when shRNAs are expressed at high levels in CNS neurons, they trigger an adverse tissue response leading to neuronal degradation.