Feline Lindhout

3 74 Camacho et al. 2013; Otani et al. 2016; Sousa et al. 2017). Thus, the profound slower development of human iPSC-derived neurons increases the temporal resolution to study axonal processes (Espuny-Camacho et al. 2013; Otani et al. 2016 Linaro, 2019 #9). Altogether, this emphasizes the relevance of studying centrosome functions during axon development in human iPSC-derived neurons as a model system. In this study, we used a multidisciplinary approach, by combining human iPSC-derived neuron cultures with live-cell imaging, electrophysiology and mass spectrometry analysis, to examine the role of centrosomes during early axon development. We found that centrosomes display microtubule organizing functions during axon specification, and this function is gradually lost during further axon development. Moreover, Trim46 localization shifts from a pericentriolar region to the AIS during neuron development, coinciding with the developmental decline of centrosomal microtubule organizing functions. Differentiation of centriole-depleted neuronal stem cells (NSCs) results in various axonal developmental defects, including immature action potential firing, mislocalization of Trim46 proteins, growth cone perturbations, and impaired axonal microtubule remodeling. Together, these data imply that centrosomes mediate microtubule remodeling during axon specification in human iPSC-derived neurons, which is necessary for correct axon formation during further development. RESULTS Centrosomes display microtubule organizing functions during axon specification We first examined whether centrosomes display microtubule organizing functions during axon specification in human neurons, by testing their potential to nucleate microtubules. To address this, we determined the endogenous levels of centrosomal γ-Tubulin, an essential microtubule nucleating protein, at different developmental stages of hiPSC-derived neurons. The neurodevelopmental stages were defined as follows: stage 1 (day 0) as Ki67-positive NSCs; stage 2 (day 7) as differentiated unpolarized MAP2-positive neurons with Trim46- negative processes; stage 3 (day 12) as differentiated polarized MAP2-positive neurons with Trim46-positive axons (Fig 1A,B). We identified centrosomes with centriole marker Centrin, and quantified the intensity levels of γ-Tubulin co-localizing with Centrin (Fig 1A,B). We found that the γ-Tubulin levels at centrosomes were consistently high in stage 1 and stage 2 neurons, and markedly reduced by ~50% in stage 3 neurons, consistent with previous findings in dissociated rat neurons (Fig 1A-C) (Stiess et al. 2010). These results suggest that centrosomes display microtubule organizing functions during the early developmental stages of human iPSC-derived neurons, i.e. during axon specification, but not during later stages.