During human brain development, trillions of synapses are formed during the highly complex yet deliberate establishment of neuronal connectivity. When this intricate process goes awry, numerous developmental and neurodegenerative disease states can manifest, including epilepsy, mental retardation, autism, Alzheimer’s, Parkinson’s, and Huntington’s disease. Despite the astonishing number and functional importance of synaptic connections, surprisingly little is known about where central nervous system synapses are established or the cellular mechanisms that guide synapse formation or continued function. Recent insights into the regulation of axonal microtubule motors provide a link to the cytoskeletal foundation required for local presynaptic vesicle delivery, a requisite for appropriate synapse function. KIF1A, a highly-processive, neuron-specific kinesin-3 motor protein, has been shown to preferentially detach from GTP-rich microtubule ends, which are present at presynaptic sites and thus promote the delivery of synaptic cargos by KIF1A. The question of how the microtubule network is locally regulated to facilitate presynaptic KIF1A off-loading remains unexplored. Here, I propose to test the hypothesis that polyglutamylation and spastin activity act in tandem to functionally distinguish microtubules along the axon to establish and maintain synapses. Previous work has revealed that spastin acts to sever microtubules to expand their mass, that polyglutamylation level dictates spastin activity, and that spastin co-localizes with presynaptic vesicles, making these factors excellent candidates for presynaptic microtubule regulators. In Aim 1, I will directly test the consequences of polyglutamylation and spastin activity on KIF1A behavior using reconstituted in vitro single molecule assays. Through the strategic use of minimal systems, this aim will untangle the complexity of microtubule network regulation and establish how two important factors, polyglutamylation and spastin, preferentially modulate KIF1A activity. Aim 2 will explore the in vivo role of polyglutamylation and spastin on the formation and maintenance of presynaptic sites along the axon in neurons derived from human pluripotent stem cells. This aim will characterize the location of polyglutamylation and spastin in relation to GTP-rich microtubules and presynaptic vesicles with high spatial resolution as well as functionally assess their role in synapse generation and maintenance by genetically disrupting the enzymes.