Molecular mechanisms of human presynapse assembly - role of liprin-alpha proteins in synapse organization and neurological disease

Abstract

Synapses are the essential transmission units of neuronal circuits and represent the cornerstone of our unique cognitive abilities. During synapse formation, contact between two neurons induce the assembly of pre- and postsynaptic compartments. On the presynaptic side, this involves the recruitment of intracellular proteins to form an active zone protein network that precisely orchestrates clustering of synaptic vesicles and their tightly controlled release. Cell-adhesion receptors present at the synapse, for example members of the neurexin (NRXN) and leukocyte common antigen-related protein tyrosine phosphatase receptor (LAR-PTPR) families, are thought to contribute to synapse assembly, but how extracellular interactions translate into downstream intracellular events remains unknown. Defects in synapse formation and function are involved in multiple neurodevelopmental and psychiatric disorders. Understanding how synaptic connections assemble is thus a central issue in neuroscience but, despite being extensively studied, the underlying molecular mechanisms have remained elusive. This thesis aimed at defining the contribution of the liprin-alpha family of scaffolding proteins in presynapse formation and neurological disease, by use of human stem cell-derived neuronal models. In paper I, we generated gene-edited human quadruple knockout neuronal models lacking all liprin-alpha isoforms and found that emergent synaptic connections are still formed but subsequent recruitment steps of synaptic components, are completely ablated, resulting in disruption of synaptic transmission. We thus identify liprin-alpha proteins as central master organizers of human presynapse assembly, bridging cell-adhesion receptors to downstream recruitment steps. In paper II, we set up a streamlined targeting system for the rapid generation of gene-edited human pluripotent stem cell lines integrating adeno-associated virus and CRISPR-Cas9 technologies. In paper III, we developed a glia-free protocol for the differentiation of human pluripotent stem cells into mature cortical glutamatergic neurons with preserved synchronous network synaptic activity. Finally, in paper IV we sought to characterize the role of liprin-alpha proteins during early neurodevelopment in vitro, uncovering a putative impact of liprin-alpha functions on the adherens junction of neuroepithelial progenitor cells. In conclusion, this thesis provides the first example of hierarchical protein recruitment in mammalian synapse assembly. Our findings may have implications for both the fundamental understanding of how synapses molecularly assemble and the processes underlying synaptic dysfunction in human disease.