Feline Lindhout

3 Centrosome-mediated microtubule remodeling during axon formation in human iPSC-derived neurons 73 Centrosomes, the main microtubule organizing center (MTOC) in most animal cells, are essential for organizing the microtubule network in unpolarized neurons (Tsai and Gleeson 2005; Stiess et al. 2010; Meka, Scharrenberg, and Calderon de Anda 2020). These small, membrane-less and centrally-localized organelles are composed of two centrioles surrounded by the pericentriolar material (PCM) (Moritz et al. 2000).The majority of the microtubules are typically nucleated from γ-Tubulin Ring Complexes (γTuRCs) embedded in the PCM (Moritz et al. 2000). During neuronal development, centrosomes gradually lose their function as MTOC as they transform into cilia, major signalling hubs in polarized cells (Stiess et al. 2010; Ishikawa and Marshall 2011). In dissociated rodent neurons, this process was reported to occur during axon development, but the exact temporal relation between axon specification and the declining MTOC function of centrosomes is unclear (Stiess et al. 2010). The importance of centrosome function in early neurodevelopment is illustrated by the increasing number of identified mutations in centrosomal proteins causing microcephaly and other neurodevelopmental disorders (Nano and Basto 2017). However, the precise function of centrosomes as MTOC for different processes of early axon development is still under debate. Progress in understanding the role of centrosomes during axon specification has been hindered due to a number of technical challenges. In particular, centrosomes are found to display different functions in neurodevelopment in different species, resulting in conflicting findings. This is mostly illustrated by the poor recapitulation of human neurodevelopmental disorders caused by centrosome dysfunction in Drosophila and mice, whereas ferrets robustly model these diseases (Basto et al. 2006; Castellanos, Dominguez, and Gonzalez 2008; Pulvers, Bryk, Fish, Wilsch-Brauninger, et al. 2010; Pulvers, Bryk, Fish, Wilsch- Bräuninger, et al. 2010; Johnson et al. 2018). Axon outgrowth can also be differently affected by centrosomes, as this process is perturbed with centrosome dysfunction in mice and peripheral axons of zebrafish, but not in dissociated rodent neurons and central axons of zebrafish (Stiess et al. 2010; de Anda et al. 2010; Andersen and Halloran 2012). The molecular mechanisms that underlie the observed species-specific differences remain largely unknown. Another technical challenge is presented by dissociated rodent neurons in culture, which are classically used to study axon developmental processes, as they likely undergo repolarization after being polarized in vivo rather than de novo polarization (Barnes and Polleux 2009). Together, this highlights the importance of studying the role of centrosomes in de novo polarization using human neurons. The development of human induced pluripotent stem cells (iPSCs) now enables studying the molecular mechanisms that drive the transition of an unpolarized human neuronal stem cell to a polarized human neuron (Lancaster et al. 2013). An additional important feature of human neurons is their significant protracted development, which allows for a more detailed investigation of the temporal processes underlying neuronal polarity (Otani et al. 2016; Linaro et al. 2019). To illustrate this, neurogenesis occurs after ~1 week in rodents, whereas this takes about ~3 months in humans, both in vivo and in vitro (Shi, Kirwan, and Livesey 2012; Espuny-