JNK-Interacting Protein 3 Mediates the Retrograde Transport of Activated c-Jun N-Terminal Kinase and Lysosomes

Abstract

Retrograde axonal transport requires an intricate interaction between the dynein motor and its cargo. What mediates this interaction is largely unknown. Using forward genetics and a novel in vivo imaging approach, we identified JNK-interacting protein 3 (Jip3) as a direct mediator of dynein-based retrograde transport of activated (phosphorylated) c-Jun N-terminal Kinase (JNK) and lysosomes. Zebrafish jip3 mutants ( jip3 ^nl7 ) displayed large axon terminal swellings that contained high levels of activated JNK and lysosomes, but not other retrograde cargos such as late endosomes and autophagosomes. Using in vivo analysis of axonal transport, we demonstrated that the terminal accumulations of activated JNK and lysosomes were due to a decreased frequency of retrograde movement of these cargos in jip3 ^nl7 , whereas anterograde transport was largely unaffected. Through rescue experiments with Jip3 engineered to lack the JNK binding domain and exogenous expression of constitutively active JNK, we further showed that loss of Jip3–JNK interaction underlies deficits in pJNK retrograde transport, which subsequently caused axon terminal swellings but not lysosome accumulation. Lysosome accumulation, rather, resulted from loss of lysosome association with dynein light intermediate chain (dynein accessory protein) in jip3 ^nl7 , as demonstrated by our co-transport analyses. Thus, our results demonstrate that Jip3 is necessary for the retrograde transport of two distinct cargos, active JNK and lysosomes. Furthermore, our data provide strong evidence that Jip3 in fact serves as an adapter protein linking these cargos to dynein.

Author Summary

To form and maintain connections, neurons require the active transport of proteins and organelles between the neuronal cell body and axon terminals. Inhibition of this “axonal” transport has been linked to neurodegenerative diseases. Despite the importance of this process, to date there was no vertebrate model system where axonal transport could be studied in an intact animal. Our study introduces zebrafish as such a model and demonstrates its power for the analysis of axonal transport. We used this system to 1) initiate a genetic screen to find novel mediators of axonal transport; 2) develop in vivo imaging strategies to visualize axonal transport in real time in the intact animal; and 3) discover, using these methods, that JNK interacting protein 3 (Jip3) is required for the transport of two cargos, a kinase and lysosomes, from axon terminals to the cell body (retrograde transport). In the absence of Jip3, these cargos accumulate and axon terminals become dysmorphic, though the retrograde transport of other cargos is normal. Interestingly, abnormal localization of these cargos has been linked to axonal disease states, but our work is the first to identify a specific adapter protein necessary for their transport from axon terminals.