Spastin locally amplifies microtubule dynamics to pattern the axon for presynaptic cargo delivery

Abstract

Neurons rely on long-range trafficking of synaptic components to form and maintain the complex neural networks that encode the human experience. With a single neuron capable of forming thousands of distinct en passant synapses along its axon, spatially precise delivery of the necessary synaptic components is paramount. How these synapses are patterned, and how efficient delivery of synaptic components is regulated, remains largely unknown. Here, we reveal a novel role for the microtubule severing enzyme spastin in locally enhancing microtubule polymerization to influence presynaptic cargo pausing and retention along the axon. In human neurons derived from induced pluripotent stem cells (iPSCs), we identify sites stably enriched for presynaptic components, termed protosynapses, which are distributed along the axon prior to the robust assembly of mature presynapses apposed by postsynaptic contacts. These sites are capable of cycling synaptic vesicles, are enriched with spastin, and are hotspots for new microtubule growth and synaptic vesicle precursor (SVP) pausing/retention. Disruption of neuronal spastin, either by CRISPRi-mediated depletion or transient overexpression, interrupts the localized enrichment of dynamic microtubule plus ends and diminishes SVP accumulation. Using an innovative human heterologous synapse model, where microfluidically isolated human axons recognize and form presynaptic connections with neuroligin-expressing non-neuronal cells, we reveal that neurons deficient for spastin do not achieve the same level of presynaptic component accumulation as control neurons. We propose a model where spastin acts locally as an amplifier of microtubule polymerization to pattern specific regions of the axon for synaptogenesis and guide synaptic cargo delivery.