Feline Lindhout

150 5 Axons are unique structures that are fundamental for information processing in neuronal networks, as they facilitate neurotransmission and maintain neuronal polarity. To carry- out these specialized functions, axons adopt extreme morphologies and characteristic substructures during development. The work in this thesis features new molecular insights in axon formation as well as axon functioning ( Chapter 2, 3 and 4 ). To summarize, in Chapter 2 a new axon developmental stage is identified in human neurons, which reveals that axon formation follows a global distal-to-proximal reorganization in the developing axon. The formation of an axon is driven by extensive remodeling of the microtubule cytoskeleton network, and Chapter 3 demonstrates that this process critically relies on centrosome function. Chapter 4 focuses on the functioning of mature axons, and unravels a new molecular interplay between the endoplasmic reticulum (ER) and the synaptic vesicle cycle at presynaptic sites. The implications of these findings are largely discussed in their corresponding chapters. This chapter primarily focuses on discussing the key findings of this thesis in a broader context, and provides future research questions and perspectives. Novel insights in axon formation from human neurons Axondevelopment isahighlycoordinatedmultistepprocess, and theonset of axondevelopment marks the first step in establishing neuronal polarity. Most insights in the molecular processes underlying axon development are obtained from non-human model systems, and in particular from the classical dissociated rodent neuron cultures (Dotti, Sullivan, and Banker 1988). However, it remains to be established to what extent the axon developmental processes identified in non-human species are recapitulated in humans. Moreover, axon development in cultured dissociated rodent neurons likely entails a repolarization event rather than de novo polarization, as these dissociated neurons in culture are obtained from yet polarized neurons in vivo (Dotti, Sullivan, and Banker 1988). Chapter 2 and 3 address new biological questions regarding axon development in human neurons, using human induced Pluripotent Stem Cells (iPSC)-derived neuron cultures, which has resulted in new insights and updated views on axon developmental processes (Figure 1) (Lancaster et al. 2013). An important hallmark of human iPSC-derived neurons that significantly contributed in obtaining these new insights, is their profound longer developmental trajectory compared to other species (Dotti, Sullivan, and Banker 1988; Petanjek et al. 2011; Shi, Kirwan, and Livesey 2012; Espuny- Camacho et al. 2013; Nicholas et al. 2013; Otani et al. 2016; Sousa et al. 2017; Linaro et al. 2019) (Chapter 2) . These species-specific differences in timing of neurodevelopment are consistently observed, also in more closely-related primate species such as humans and macaques (Otani et al. 2016). In fact, this prolonged human-specific developmental timing is even maintained when human neurons are transplanted in rodent brains or when human neurons are co-cultured with macaque neurons (Espuny-Camacho et al. 2013; Nicholas et al. 2013; Otani et al. 2016; Linaro et al. 2019). Together, this implies that neurodevelopmental timing in different species is orchestrated by robust and cell-intrinsic mechanisms. What controls this internal molecular clock in neuronal cells? It would be interesting to direct future studies in addressing this question, as mechanistic insights are still lacking at this point.