Project Summary The long-term goal of this project is to define molecular mechanisms that govern the long distance transfer of protein-based signals in axons. Retrograde (axon-to-soma) signals are critical to activate transcriptional programs both during neurodevelopment and following nerve injury, while continuous anterograde (soma-to- axon) supply of `axon survival factors' is essential to maintain distal axon integrity. We and others have found that key proteins that convey these retrograde and anterograde signals are modified with the lipid palmitate, which facilitates their trafficking on axonal vesicles. In particular, experiments in the first cycle of funding revealed that retrograde signaling by Dual Leucine-zipper Kinase (DLK, an upstream activator (a `MAP3K') of Mitogen-activated Protein Kinase (MAPK) pathways) critically requires palmitoylation. We now hypothesize that palmitoylation more broadly controls several distinct aspects of axonal signaling. The first Aim will focus on palmitoylation of JNK family MAPKs, which are key `effector' kinases downstream of DLK and other MAP3Ks. We will determine whether palmitoylation of the neural-specific JNK3 is required for Wallerian degeneration of distal axons, and whether JNK3 phosphorylates palmitoylated axon survival factors, triggering their degradation via a novel phospho-dependent mechanism. Aim 2 will focus on Rap2, a novel palmitoylated regulator that lies upstream of DLK, and will determine whether Rap2 and its palmitoylation are broadly required for DLK-dependent retrograde signaling. Aim 3 will assess whether the unique reversibility of palmitoylation, compared with other protein-lipid modifications, is used to facilitate `sushi belt transport' whereby the key axon survival factor NMNAT2 undergoes palmitoylation-dependent anterograde trafficking on vesicles and is then locally depalmitoylated to increase its enzymatic and axo-protective activity. The proposed research will define new cellular and molecular roles for palmitoylation in axonal protein trafficking and signaling and will provide key insights into how responses to axonal damage are coordinated and controlled. Results of our study may also reveal broader principles of axonal protein transport and signaling, in turn increasing our understanding of a range of neurodegenerative disorders in which these processes are impaired.