Feline Lindhout

3 80 Centriole loss perturbs axonal targeting of Trim46 We next asked whether these prematurely differentiated neurons upon Centrinone-B treatment follow normal developmental timing and grow functional axons. To investigate the role of centrosomes during axon specification, we assessed if the development of early-stage axons was affected by centriole loss. An important aspect of early-stage axon development is the specific sorting of axonal proteins, including AIS proteins such as Trim46 and AnkG. Thus, we tested if the axon-specific localization of Trim46 and AnkG was affected by Centrinone-B induced centriole removal. We divided the neurons treated with Centrinone-B into subpopulations containing 2 or <1 centriole(s), as defined by Centrin immunostaining (Fig 2C). Cells were co-immunostained with Trim46 and AnkG to quantify the number of cells containing a Trim46-positive and/or AnkG-positive process (Fig 2C). We observed a marked ~50% reduction of cells with a Trim46-positive process in the subpopulation containing <1 centriole(s) compared to cells still containing 2 centrioles, whereas no changes were observed for AnkG (Fig 2D,E). Consistently, cells containing <1 centriole(s) showed reduced Trim46 co-localization at AnkG-positive axonal structures (Fig 2F). Together, these data imply that centrosomes are required for the targeting of Trim46, but not AnkG, to axons during early stages of neuronal development. Centriole loss leads to immature action potential firing The axonal targeting of Trim46 and AnkG is required to assemble the AIS, the highly specialized structure essential for mature and efficient AP firing. Thus, we assessed if the observed differential effects on axon protein targeting upon centriole depletion correlate with changes in AP properties. We performed whole-cell patch clamp recordings of control or Centrinone-B treated neurons of 7-14 days, which coincides with early axon development. To measure neuronal excitability, we determined the number of APs fired with increasing somatic current injection (steps of 5 pA; 400 ms) (Fig 2G, Fig S2F). In Centrinone-B treated cultures, ~17% of neurons did not fire APs, whereas this was only ~3% in control cultures (Fig 2H). Of the firing neurons, there were less Centrinone-B treated neurons that fired multiple APs compared to control. Neurons that did not fire APs did generate small peaks upon current stimulation, indicating the opening of sodium channels, but no positive feedback to rapidly increase the membrane potential as is characteristic of APs. In addition, neurons treated with Centrinone-B did not display a progressive maturation of AP properties from day 10 to day 14, as was observed in control neurons (Fig 2I). APs fired by Centrinone-B treated neurons appeared more immature, as they were wider, had smaller amplitudes and smaller after-hyperpolarizations (Fig 2J-L, S2G,H). In Centrinone-B treated neurons the input resistance was also higher, but membrane potential did not differ from control (Fig S2I,J). AlthoughAP threshold was not affected by Centrinone-B treatment (Fig 2L), maximum sodium currents were significantly smaller in Centrinone-B treated neurons (Fig 2M). Together, the electrophysiology recordings from Centrinone-B treated neurons show more immature AP firing and reduced sodium currents, thereby highlighting the functional relevance of centrosome-mediated control mechanisms during early stages of neuronal development.