|dc.description.abstracteng||Seminal methodological advancements in the field of human genetics now allow for low-cost sequencing of complete coding genomes, and rich sources of corresponding data now exist for healthy and patient cohorts, allowing for the identification of possibly pathogenic variations. However, lagging behind is the identification of mechanisms by which such variations lead to disease, both at the molecular and cellular levels. Multiple genetic studies of neurodevelopmental and neuropsychiatric disorders now point to a central role of synaptic transmission in the etiology of such disorders. This project aims to shed light on molecular mechanisms and synaptic transmission abnormalities underlying a novel disorder of the presynaptic terminal – associated with variations in the UNC13A gene that translates into the Munc13-1 protein.
Munc13-1 is a large, multi-domain protein absolutely essential for priming synaptic vesicles (SV) to the active zone plasma membrane. The absence of Munc13-1 in mice leads to severely compromised synaptic transmission that is lethal. Evidence also exists pointing to the fact that Munc13-1 is pivotal for nervous system function in humans. Recently, the first patient with a de novo variant in the Munc13-1 protein, was described. The patient phenotype was characterized by a dyskinetic movement disorder, developmental delay and autism. The patient variant, P814L, leads to a gain of function of both excitatory and inhibitory synaptic transmission – neurons expressing this variant exhibited an increase in the basal SV release probability and aberrant patterns of short-term synaptic plasticity during trains of action potentials. Following this publication, a collaboration with the group of Prof. Dr. med. Anita Rauch from the Institute of Medical Genetics at the University of Zurich was initiated. This research group excels in the collection of patient data and in in-depth genetic analysis of human material. By now, around 40 patients were identified, who carry de novo or biallelic variants in the UNC13A gene, presenting with variable neurological and neuropsychiatric conditions. The patient variants are distributed over the entire Munc13-1 protein sequence and in every functional domain.
In this thesis, I am presenting electrophysiological and immunocytochemistry data analysing the functional consequences of five patient variants, characterized using the mouse hippocampal autaptic neuron culture system. One variant mirroring the phenotype of the previously reported P814L mutation confirms the reproducibility of our experimental system and its ability to accurately capture a phenotypic-functional relationship. Beyond that, I distinguish two distinct mechanisms in disease. One group of patients accumulating in the highly conserved C2B-MUN-Linker domain elicits a gain of function in synaptic transmission properties. In contrast, a second group of patients accumulates on the less conserved N-terminus and displays a loss of function phenotype in our electrophysiological measurements. My findings uncover two distinct mechanisms governing disease manifestation in UNC13A patients, and emphasize the significance of genetic variants in synaptic proteins as causal for brain disorders.||de