# The Role of Intracellular Trafficking in Regulating Axon Guidance Receptors During Neuronal Circuit Formation

> **NIH NIH F31** · UNIVERSITY OF PENNSYLVANIA · 2022 · $34,090

## Abstract

Project Abstract
To form a functional nervous system, each neuron must find and connect to the appropriate synaptic targets.
Axons must often travel long distances in search of their targets, directed by a variety of secreted and cell
surface axon guidance molecules. These ligands steer axons by interacting with receptors on their growth
cones and altering cell behavior through changes in cytoskeletal architecture, cell adhesion, and transcription.
The complex trajectories that axons take to reach their destinations are often broken up into smaller segments,
which are punctuated by intermediate targets or “choice points.” In bilateral organisms, the midline is a vital
choice point for a subset of CNS neurons, called commissural neurons, whose axons must cross it to integrate
information between the left and right halves of the body. Midline crossing is necessary for normal motor and
cognitive function and defects in this vital process can result in a wide variety of neurodevelopmental disorders.
Midline crossing is controlled by a combination of attractive and repulsive cues secreted by midline glia.
Commissural neurons are initially sensitive to attractive cues such as Netrin, which draw them to the midline.
After crossing, however, they become responsive to repulsive cues such as Slit, which facilitate exit from the
midline and prevent re-crossing. Pre-crossing commissural neurons must prevent premature responsiveness
to these repulsive cues and do this, in part, by downregulating the Slit receptor Roundabout (Robo). Drosophila
downregulate Robo surface expression via the trafficking receptor Commissureless (Comm), which shunts
newly-synthesized Robo directly from the Trans-Golgi Network to the endolysosomal system and decreases
Robo protein levels. Two PY motifs in the cytoplasmic tail of Comm are necessary for its ability to perform
these tasks. As PY motifs are known binding sites for Nedd4-family HECT ligases, this strongly suggests that
Comm downregulates Robo by interacting with intracellular ubiquitination machinery. The precise mechanistic
relationship between Comm and ubiquitin ligases, however, has not been explored in great detail. In this
proposal, we seek to test the hypothesis that Comm acts as an adaptor protein that brings E3 ligases into
close proximity to Robo, and that ubiquitination of Robo targets it to the endolysosomal system for degradation.
In aim one, we seek to elucidate the requirement for ubiquitin to traffic the Comm-Robo complex to
endo/lysosomes, facilitate Robo degradation, and promote midline crossing. In aim two, we will investigate the
biochemical and genetic interactions between Comm and the three Drosophila Nedd4 family HECT ligases
(Nedd4, Su(dx), and Smurf). In summary, the proposed work will provide deeper insight into the mechanisms
regulating surface expression of axon guidance receptors during the development of neural circuits.

## Key facts

- **NIH application ID:** 10318151
- **Project number:** 5F31NS115345-03
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Kelly Gale Sullivan
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $34,090
- **Award type:** 5
- **Project period:** 2019-12-01 → 2022-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10318151

## Citation

> US National Institutes of Health, RePORTER application 10318151, The Role of Intracellular Trafficking in Regulating Axon Guidance Receptors During Neuronal Circuit Formation (5F31NS115345-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10318151. Licensed CC0.

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