Diversity and Specificity of Actions of Slit2 Proteolytic Fragments in Axon Guidance

TLDR

Slit proteins, particularly Slit2, are secreted guidance cues that bind Robo receptors to regulate axon guidance, neuronal migration, and midline crossing, and are processed into N‑ and C‑terminal fragments while the full‑length form also exists. The study aimed to investigate the functional activities of Slit2 fragments by engineering mutant and truncated versions representing N‑, C‑, and full/uncleavable forms. They generated mutant and truncated Slit2 constructs (N‑, C‑, and full/uncleavable) and tested their binding to Robo receptors and effects on axon guidance, growth‑cone collapse, and branching in collagen‑gel cultures of olfactory bulb and dorsal root ganglia axons. Only the N‑terminal fragment and the uncleavable full‑length Slit2 bound Robo receptors, repelled olfactory‑bulb axons, and induced growth‑cone collapse, while only the N‑terminal fragment promoted dorsal‑root‑ganglia axon branching, an effect blocked by the uncleavable form, demonstrating fragment‑specific guidance and growth‑promoting activities.

Abstract

The Slits are secreted proteins that bind to Robo receptors and play a role in axon guidance and neuronal migration. In vertebrates, Slit2 is a major chemorepellent for developing axons and is involved in the control of midline crossing. In vivo , Slit2 is cleaved into 140 kDa N-terminal (Slit2-N) and 55–60 kDa C-terminal (Slit2-C) fragments, although the uncleaved/full-length form can also be isolated from brain extract. We explored the functional activities of Slit2 fragments by engineering mutant and truncated versions of Slit2 representing the N-, C-, and full/uncleavable (Slit2-U) fragments. Only Slit2-N and Slit2-U bind the Robo proteins. We found that in collagen gel, olfactory bulb (OB) but not dorsal root ganglia (DRG) axons are repelled by Slit2-N and Slit2-U. Moreover, only Slit2-N membranes or purified protein-induced OB growth cones collapse. Finally, we found that only recombinant Slit2-N could induce branching of DRG axons and that this effect was antagonized by Slit2-U. Therefore, different axons have distinct responses to Slit2 fragments, and these proteins have different growth-promoting capacities.