Feline Lindhout

154 5 is plausible that this process is mediated by different local intracellular signaling pathways during the transition from stage 2 to stage 3 neurons (Conduit, Wainman, and Raff 2015). Alternatively, the presence of these proteins at both centrosomes and axons perhaps reflect different isoforms of these proteins, which might be differentially expressed in stage 2 and stage 3 neurons. To this end, it is interesting to highlight that humans contain more Trim46 isoforms with yet unknown functions compared to other species, and that the centrosome location of Trim46 is primarily observed in human cells (Chapter 3) . In conclusion, the functional relevance of centrosomes during the onset of axon development is emerging, and future studies are necessary to unravel the underlying molecular mechanisms. Emerging roles of the endoplasmic reticulum for presynaptic function Presynaptic boutons are crucial sites for neurotransmitter release. This is facilitated by the highly controlled synaptic vesicle cycle, in which neurotransmitter-containing synaptic vesicles proceed through cycles of exocytosis and retrieval at presynaptic sites. Many studies in the past decades focused on investigating this synaptic vesicle cycle, which has greatly advanced our understanding of the underlying molecular mechanisms. However, the contribution of abundant presynaptic organelles, such as the ER and mitochondria, in synaptic vesicle recycling is much less explored. The ER and mitochondria present throughout axons, including presynaptic sites, have adopted unique structures. The axonal ER network is marked by extremely narrow ER tubules, which form local networks of small cisternae at presynaptic boutons (Yalcin et al. 2017; Wu et al. 2017; Terasaki 2018). Axonal mitochondria also display a distinctive morphology marked by short and punctate structures, thereby differing from the long and tubular structures observed in dendrites and most other cell types (Chicurel and Harris 1992; Li et al. 2004; Popov et al. 2005; Dickey and Strack 2011; Kasthuri et al. 2015). Mitochondria at presynaptic sites occupy a large volume, up to one- third, of the presynaptic bouton (Wilhelm et al. 2014). The presence and extreme adaptations of the ER and mitochondria at presynaptic boutons and throughout axons are indicative of their relevance for presynaptic function. Indeed, the findings in Chapter 4 uncovered the importance of the ER structure and its dynamic remodeling for presynaptic function (Fig 1). Here, a new molecular control mechanism for maintaining ER continuity is identified, mediated by the interaction between ER receptor vesicle-associated membrane protein (VAMP)-associated protein B (VAPB) and VAP-interacting protein Secernin-1 (SCRN1). Perturbing ER structure and dynamics, by interfering with VAP-SCRN1 interactions, largely affected presynaptic function. More specifically, the comprised presynaptic function was marked by a reduction in synaptic vesicle recycling, a decrease in evoked Ca 2+ responses and an increase in basal Ca 2+ levels at presynaptic boutons. Together, these data imply that the dynamic ER structures are important to maintain local Ca 2+ levels and facilitate Ca 2+ influxes at presynaptic sites, thereby modulating synaptic vesicle recycling. This is consistent with the well-described key function of the ribosome-lacking smooth ER, the primary type of ER present in axons, in controlling Ca 2+ homeostasis (Yalcin et al. 2017; Wu et al. 2017; Terasaki 2018). The important molecular interplay between ER and presynaptic