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.