Feline Lindhout

General discussion 151 5 The prolonged development of human iPSC-derived neurons makes it a particularly interesting model system to study developmental processes that typically occur at relative fast rates, such as the first steps of axon development. Specifically, the process of axon formation, involving axon specification and axon initial segment (AIS) assembly, occurs after ~24-48 hours in dissociated rodent neurons, whereas this takes ~7-14 days in human iPSC-derived neurons (Dotti, Sullivan, and Banker 1988) (Chapter 2) . This stretched time window of axon development in human neurons increases the temporal resolution to study this process in more detail. As such, Chapter 2 uncovers a previously unrecognized intermediate stage during early axon development, revealing that axon formation is first initiated distally in the future axon which is next followed by proximal rearrangements (Fig 1). Specifically, this development in a distal-to-proximal fashion is observed for the reorganization towards the axon-specific microtubule network, as well as the appearance and structural assembly of AIS proteins. It remains to be explored how these distal-to- proximal rearrangements are driven mechanistically over time. As indicated by their name, AIS proteins are originally defined by their collective localization at proximal axons, where they assemble into a highly specialized AIS structure (Leterrier 2018). Thus, their distal appearance at immature axons opens up new discussions, and follow-up studies are necessary to reassess the function of AIS proteins during early axon development. In conclusion, future studies may identify more novel axon developmental processes and dissect its molecular details, and the potential human iPSC-derived neuron cultures to address this is illustrated in Chapter 2 and 3 . Centrosomes, as microtubule-organizing organelles, are important for axon formation Axon formation starts with the by accelerated outgrowth of a single neurite, the future axon. These drastic morphological changes are driven by structural rearrangements of the local microtubule cytoskeleton. Chapter 3 reveals that this process critically depends on the centrosome, the organelle acting as the main microtubule-organizing center in cells (Figure 1). Depleting neuronal stem cells of centrioles, the core components of centrosomes, results in impaired axon-specific microtubule remodeling at later stages. This is accompanied by additional axon developmental defects, including immature firing of action potentials, mistargeting of the axonal microtubule-associated protein Trim46, suppressed expression of growth cone proteins and impaired growth cone morphologies. Together, these data reveal that centrosomes are indispensable for microtubule remodeling during early axon development, which is important for setting-up the foundation for subsequent axon maturation and functioning. Previous studies reported contradicting results on the interplay between centrosomes and axon formation, resulting in an ongoing debate on the function of centrosomes during this developmental process (Zmuda and Rivas 1998; de Anda et al. 2005; Stiess et al. 2010; Meka, Scharrenberg, and Calderon de Anda 2020). Accumulating evidence revealed that centrosomes are positioned at sites were the new axon emerges, indicating that a possible mechanistic link between localized centrosome