KIF1A ‐Mediated Axonal Transport of Netrin‐1 in iPSC ‐Derived Human Forebrain Neurons

Netrin‐1 is a secreted laminin‐like protein that functions as a guidance cue for axon pathfinding in development. Netrin‐1 also modulates synapse formation in developing neurons and long‐term potentiation in mature neurons. Netrin‐1 is expressed in human neurons; however, its intracellular distribution and trafficking have not been assessed. Moreover, there is a genetic association in Caenorhabditis elegans between UNC‐6/netrin‐1 and UNC‐104/KIF1A, a kinesin‐3 motor protein required for axonal transport. We characterized the endogenous expression of netrin‐1 via immunocytochemistry and demonstrated it is localized to both the axon and dendrites of induced pluripotent stem cell (iPSC)‐derived human forebrain neurons. Next, live‐cell imaging of netrin‐1‐mRFP revealed rapid, bidirectional axonal transport, comparable to that of other cargos undergoing microtubule‐based trafficking in vertebrate neurons. To identify the netrin‐1 vesicle population, co‐localization analyses demonstrated that netrin‐1 puncta overlap with the dense‐core vesicle marker Chromogranin A. Netrin‐1‐mRFP and Chromogranin A‐eGFP also undergo co‐transport within the axon. Finally, we demonstrated a role for the kinesin‐3 motor protein, KIF1A, in Netrin‐1 transport by demonstrating that KIF1A‐associated neurological disease (KAND) patient variants significantly reduce netrin‐1 axonal transport.