Regulation of axon guidance receptor trafficking in the developing mammalian central nervous system

PROJECT SUMMARY
Determining how neurons are assembled into functional circuits will provide insight into developmental
disorders of the nervous system and may suggest therapeutic approaches to promote nerve regeneration. To
navigate to their correct targets, axons must modulate their responses to extracellular cues, and regulated
intracellular protein trafficking plays a pivotal role in this process. For example, commissural axons cross the
midline despite the presence of repellant ligands in order to establish connections that are essential for
coordinated motor behavior. In Drosophila, the endosomal protein Commissureless (Comm) prevents
commissural axons from prematurely responding to the repellant Slit, by inhibiting surface expression of the
Slit receptor Roundabout1 (Robo1). In mammals, Robo receptors are also negatively regulated in commissural
axons prior to midline crossing, but the mechanisms are unknown. Unlike Slit and Robo, comm is not
conserved in vertebrates; however, our preliminary data indicate that the vertebrate Nedd-4 interacting proteins
(Ndfip1 and Ndfip2) can act analogously to Comm to regulate the trafficking and stability of human Robo
receptors in vitro, and that loss of Ndfip1or Ndfip2 function in vivo in mice results in increased expression of
Robo receptors and defects in axon guidance. We will test the hypothesis that Ndfip proteins control axon
guidance in the developing brain and spinal cord by recruiting Robo receptors to endosomes and triggering
their degradation through interactions with Nedd-4 E3 ubiquitin ligases. In aim 1, we will use molecular, cell
biological and biochemical approaches to: 1) determine whether Ndfip proteins exhibit differential effects on
intracellular trafficking of Robo receptors or other axon guidance receptors, 2) delimit the sequences that are
necessary and sufficient to mediate interactions between Ndfip proteins and Robo family receptors, 3)
characterize the role of HECT E3 ligase activity on receptor trafficking and 4) identify the specific Nedd4 family
ligase(s) that is required for Robo receptor regulation. Aim 2 will explore the embryonic expression patterns
and in vivo requirements for Ndfip proteins during commissural axon guidance by examining the trajectory of
commissural axons in Ndfip1 and Ndfip2 single and double mutants, using 1) immunofluorescence for pre and
post-crossing commissural axon markers, and 2) unilateral lipophilic dye tracing experiments. In addition, we
will generate conditional knockouts of Ndfip1, Nedd4-1 and Nedd4-2 using Cre-lines specific for commissural
neurons to investigate requirements for Nedd4-1 and Nedd4-2 in spinal commissural axon guidance. Aim 3 will
assess the in vivo links between Ndfip proteins and Robo receptors by 1) testing whether neurons cultured
from Ndfip mutants exhibit altered repulsive responses to exogenously added Slit proteins and 2) examining
genetic interactions between Ndfip and Robo mutants. Finally, a biochemical screen will be conducted to
identify novel substrates of Ndfip proteins.

Grant Number: 5R01HD105946-05
NIH Institute/Center: NIH
Principal Investigator: Greg Bashaw