Summary of the main findings
Regulated secretion of signalling molecules from SVs and DCVs is the fundamental manner by which neurons communicate. In non-neuronal cells and nematode motorneurons, DCVs may undergo fusion reactions to specific intracellular compartments as part of their maturation process. In mammalian neurons, little is known about the pathways that control the generation of DCVs. In Chapter 2, the proteins Vti1a/b, which control DCV numbers in chromaffin cells, were studied as candidates to mediate DCV biogenesis in mammalian neurons. We observed that not only were the numbers of DCVs reduced in Vti1a/b double knockout (DKO) neurons, but also the density of synapses and the levels of proteins that mediate synaptic secretion. This translated into a severe reduction in the secretion capacity. The delivery of DCV-cargo and synaptic secretion proteins into axons was impaired. Proteins accumulated at the Golgi. The non-homogeneous distribution of DCV-cargo within the Golgi was lost. These defects were equally restored by expression of Vti1a or 1b. Furthermore, Golgi cisternae were distended in DKO neurons and the transit time of two different cargoes through this organelle was increased. Finally, retrograde transport to the Golgi, but not anterograde, was impaired. We conclude that Vti1a/b support regulated secretion by sorting proteins at the Golgi.
Dysregulation of SV and DCV pathways is associated with many psychiatric disorders. Cell reprogramming allows to study these pathways in patient-derived neurons. However, these studies are limited by the unknown maturation stage of the SV pathway and the little insight available on the DCV pathway in human reprogrammed neurons. Thus, we aimed to implement tools to characterize the DCV pathway and measure the maturation of synaptic transmission in human neurons. In Chapter 3, we defined a protocol to differentiate cortical neurons from human induced-pluripotent stem cells, resulting in balanced and reproducible networks of active glutamatergic and GABAergic neurons suitable for functional studies at cellular resolution. In Chapter 4, using the protocol defined in chapter 3, we developed quantitative assays to assess DCV trafficking and secretion at single-vesicle resolution and monitor evoked SV secretion. We show that DCV trafficking and secretion in human neurons is similar to rodents. In addition, while the DCV secretory pathway reaches its maximum capacity, the SV secretory pathway is still developing after 50 days in-vitro.