Feline Lindhout

3 88 of how centrioles mediate axonal microtubule rearrangements in human iPSC-derived neurons remains largely unclear. Developmental decline of centrosomal microtubule nucleation is a controlled process occurring after axon specification (Stiess et al. 2010). Perturbing this controlled timing by removing centrioles prior to axon specification may result in a differentially organized microtubule network in young neurons. Interestingly, a relative increase in acentrosomal microtubules was found in epithelial cells subjected to Centrinone-B induced centriole loss (Martin et al. 2018). We speculate that premature centriole loss may promote acentrosomal microtubule nucleation in axons and thereby increase minus-end out microtubules. Alternatively, centrosomes were also found to play a role in non-microtubule functions that are relevant for neurodevelopment, such as intracellular signaling, protein homeostasis and organizing the actin cytoskeleton (Conduit, Wainman, and Raff 2015; Farina et al. 2016; Vora and Phillips 2016; Meka, Scharrenberg, and Calderon de Anda 2020). However, it remains unknown to what extent these functions contribute to axon formation. Altogether, our data show that centrosomes are critical for setting-up the correct microtubule organization during axon specification, which is important for subsequent microtubule remodeling in growing axons. Centrosome dysfunction results in premature differentiation and repressed axon development Neurodevelopment is orchestrated by a highly temporal controlled sequence of events. Centrosome dysfunction significantly perturbs this coordinated timing, resulting in microcephaly disorders (Nano and Basto 2017). These reduced brain sizes are mostly attributed to a reduction of the NSC pool, due to premature differentiation and cell death (Nano and Basto 2017). Here we show that NSCs prematurely differentiate into neurons when centrosome function is impaired, consistent with previous reports in human iPSC- derived cerebral organoids (Lancaster et al. 2013). However, previous studies did not reveal whether these prematurely differentiated neurons subsequently follownormal developmental timing and are able to grow functional axons. Here we found reversed timing effects on axon development in prematurely differentiated neurons, as centriole loss perturbed microtubule remodeling during axon formation. The structural axon developmental defects were also accompanied by compromised axon function, as demonstrated by impaired AP firing and reduced sodium currents with centrosome dysfunction. Presumably, the observed functional axon defects are the result of a perturbed organization of the axonal microtubule network in centriole-depleted neurons. Setting-up the distinct microtubule organization in axons and dendrites is an important aspect of neuron polarity, and altering this process might have severe consequences for polarized cargo transport and further neurodevelopment (van Beuningen and Hoogenraad 2016). In the brain, correct timing of axonal outgrowth during neurodevelopment is key, as axon pathfinding is steered by gradients of chemical attractants or repellents that are highly spatiotemporally controlled (Stoeckli 2018). We therefore speculate that centrosome-mediated alterations in axon development may significantly affect axon targeting and neuronal innervation in vivo . To this end, it would be