Feline Lindhout

3 72 ABSTRACT Axon formation critically relies on local microtubule remodeling, and marks the first step in establishing neuronal polarity. However, the function of the microtubule-organizing centrosomes during the onset of axon formation is still under debate. Here, we demonstrate that centrosomes play an essential role in controlling axon formation in human induced pluripotent stem cell (iPSC)-derived neurons. Depleting centrioles, the core components of centrosomes, inunpolarizedhumanneuronal stemcells results invariousaxondevelopmental defects at later stages, including immature action potential firing, mislocalization of axonal microtubule-associated Trim46 proteins, suppressed expression of growth cone proteins and affected growth cone morphologies. Live-cell imaging of microtubules reveals that centriole loss prevents axonal microtubule reorganization towards the unique parallel plus- end out microtubule bundles during early development. We propose that centrosomes mediate microtubule remodeling during early axon development in human iPSC-derived neurons, thereby laying the foundation for further axon development and function. INTRODUCTION Neuronal polarity is established by a series of highly coordinated processes starting with the formation of the future axon. During axon specification, the first step of axon formation, one of the multiple unpolarized neurites of a neuron displays extenstive growth. Axonal outgrowth critically relies on local cytoskeleton reorganization and growth cone dynamics (Dotti, Sullivan, and Banker 1988; Witte, Neukirchen, and Bradke 2008). Next, the newly developed axon undergoes significant reorganization as it matures, thereby adopting axon-specific hallmarks required for its function. An essential component of mature axons is the axon initial segment (AIS), a specialized compartment at the base of the axon where specific proteins (e.g. AnkG scaffolds, microtubule-organizing protein Trim46, and voltage-gated sodium and potassium channels) assemble in a highly organized manner (Leterrier 2018; Freal et al. 2019). The AIS is crucial for maintaining neuronal polarity and generating action potentials (APs). The characteristic shaping and subsequent propagation of APs is facilitated by the local clustering of voltage-gated channels at the AIS (Kole et al. 2008). Another particularly important aspect of mature axons is their unique microtubule organization. In growing axons, the microtubule network undergoes extensive remodeling, as it shifts from a mixed microtubule polarity to a uniform plus-end out microtubule organization (Yau et al. 2016). Trim46 proteins targeted to the AIS act as regulators of these axonal microtubule rearrangements, by forming parallel microtubule bundles in proximal axons (van Beuningen et al. 2015). In contrast, dendrites contain a microtubule organization of mixed polarities, and gain additional minus-end out microtubules during development. This prominent difference in microtubule organization between axons and dendrites is essential for neuronal development and function, as it contributes to polarized cargo transport and the characteristic neuronal morphology (Baas et al. 1988; Yau et al. 2016). However, while microtubule remodeling in growing axons is important for axon specification and development, the mechanisms driving these microtubule cytoskeletal rearrangements remain largely unresolved.