Pathogenic KIF1A variants differentially disrupt axonal trafficking and impede synaptic development.

The nervous system relies on billions of neurons connected through trillions of synapses to support vital functions. Despite the importance of this synaptic network, cellular mechanisms dictating synapse formation during human neurodevelopment remain unclear. Long-distance trafficking by the microtubule motor KIF1A is crucial for synaptogenesis and downstream synapse maintenance. Mutations in KIF1A cause KIF1A-associated neurological disorder (KAND). We employed isogenic gene-edited human-induced pluripotent stem cell -derived neurons to assess the effects of disparate pathogenic mutations in KIF1A on synaptic trafficking and function. Null (p.C92*) and hypoactive (p.P305L) mutations delay neurite outgrowth, mislocalize synaptic cargoes, and decrease synapse density. Conversely, a hyperactive (p.R350G) mutation supports neurite outgrowth but causes aberrant motility of synaptic vesicle precursors and deficits in microtubule-dependent presynaptic patterning. Functional analysis of neuronal activity reveals delayed synaptic maturation in loss-of-function mutations (p.P305L, p.C92*) and precocious activity in the hyperactive p.R350G mutation. These data provide insights into how KIF1A mutations with distinct molecular-level impacts lead to significant downstream synaptic deficits in human neurons.