Proper axon outgrowth and path finding are essential for the formation of functional neuronal circuits in the developing nervous system. Coordination of different guidance cues, their receptors and intracellular signal transduction cascades eventually converges on orchestrating cytoskeleton dynamics to maneuver growth cone navigation. Netrins, a conserved family of canonical guidance cues, play an important role in axon outgrowth and guidance in the developing nervous system. The coupling of axon guidance cues, such as netrin-1, to microtubule (MT) dynamics is crucial for growth cone (GC) navigation. However, whether axon guidance signaling regulates MT dynamics directly or indirectly is unclear. Our preliminary data indicate that DCC and DSCAM interact directly with TUBB3, the most dynamic ?-tubulin isoform in neurons, and netrin-1 induces these interactions. TUBB3 is required for netrin-1- induced axon outgrowth and path finding in the developing nervous system. These results suggest that DCC and DSCAM directly couple MT dynamics in netrin-1-mediated axon attraction. Interestingly, UNC5 also interacts with TUBB3. In this study, we will determine how netrin-1 modulates microtubule dynamics through coupling of its receptors DCC, DSCAM and UNC5C with dynamic TUBB3 in attractive and repulsive signaling.
Aim1. Untangle the role of TUBB3 in coordination of netrin-1 attractive signaling. To further investigate the coordinating role of TUBB3 in netrin/DCC/DSCAM-mediated axon attractive signaling, we will examine: 1) Netrin-1 regulation of the interaction of TUBB3 with DCC and DSCAM;2) Modulation of MT dynamics by netrin-1 via the interaction of dynamic TUBB3 with DCC and DSCAM;3) The functional importance of TUBB3 in netrin attractive signaling.
Aim 2. Determine the role of TUBB3 in coordination of netrin-1 repulsive signaling. UNC5 interacts with DCC and DSCAM and these interactions mediate repulsive responses to netrin-1. Our preliminary data show that TUBB3 interacts with UNC5C, DCC and DSCAM, suggesting it may play a role in coordinating netrin-1 repulsive signaling. To further characterize this hypothesis, we will focus on studying: 1) Netrin-1 regulation of the interaction of TUBB3 with UNC5C, DCC and DSCAM;2) Modulation of MT dynamics by netrin-1 via coordination of the interaction of dynamic TUBB3 with UNC5C, DCC and DSCAM;3) The functional importance of TUBB3 in netrin-1-mediated axon repulsion.
Aim 3. Investigate the mechanistic link between netrin signaling and MT dynamics. To examine how netrin-1 directly regulates MT dynamics via TUBB3 in axon turning, we will focus on: 1) assessing tyrosine phosphorylation of TUBB3 in netrin signaling;2) tracking modulation of MT dynamics in the GC during netrin-1-mediated attraction using live cell imaging;3) tracking netrin-1- mediated MT dynamics in the GC during axon repulsion. This study should reveal how netrin-1 directly modulates MT dynamics via differential coupling of its receptors with dynamic TUBB3 in axon turning.
Many human diseases involve defects in neuronal positioning and aberrant axonal growth;including lissencephaly, double cortex, periventricular heterotopia, epilepsy, and Alzheimer's, Parkinson's disease, stroke, Spinal Muscular Atrophy (SMA), and spinal cord injury. The secreted protein netrins are prototypical guidance cues for projecting axons and migrating neurons in the developing nervous system. Studying netrin signaling will not only help us understand how the brain establishes its complex and precise neuronal circuitries during development, but also provide a basis to design new strategies for injury-related diseases, such as stroke and spinal cord injuries.