From Principles to Disease
Authors:   , Öttl Miriam 
Published: January 2025

This thesis had two general aims: 1) understanding the role of tomosyns in synaptic transmission, and 2) furthering our knowledge on specific and general disease mechanisms of STXBP1-related disorders. Chapter 2 was dedicated to uncovering tomosyn’s mode of action. A new mouse model in which both tomosyn paralogues can be conditionally removed allowed us to study the effect of complete knockout of tomosyns on synaptic transmission using a single-cell electrophysiology assay. This revealed strongly increased spontaneous as well as evoked synaptic vesicle fusion, and application of hypertonic sucrose further demonstrated that tomosyns reduce vesicular release probability by adding to the energy barrier for fusion. Tomosyn knockout could be rescued by full-length tomosyn but not by truncation mutants as well as a mutant in which the tomosyn SNARE domain was replaced by the VAMP2 SNARE domain. In addition, single molecule optical tweezer experiments showed that tomosyns interact with the template complex of Munc18-1, syntaxin-1 and VAMP2, blocking entry of SNAP25. These results together demonstrate that tomosyns do not simply replace VAMP2 and sequester the other SNARE proteins, but that they inhibit vesicle fusion by preventing the formation of the SNARE complex via the template complex. In chapter 3, we focused on the disease associated STXBP1 variant L446F. We engineered iPSCs derived from a healthy donor to introduce this mutation on both alleles, mimicking the situation in patients who are homozygous for this mutation. In contrast to wildtype controls, induced neurons harboring the mutation showed reduced depression upon multiple stimuli in electrophysiological experiments. The mutation also partially prevented binding of an antibody, as shown by immunocytochemistry and Western blot analysis. Together these results confirmed a gain-of-function of this variant on MUNC18-1 function in synaptic transmission, and indicate that protein-protein interactions may underlie this effect. In chapter 4, we compared neurons derived from multiple patients with STXBP1-related disorders to neurons derived from multiple healthy donors functionally using calcium imaging and electrophysiology, and molecularly using proteomics and immunocytochemistry. All patient neurons had reduced MUNC18-1 expression levels compared to the controls and showed disturbed network but not single-cell activity. Proteomics revealed disturbed synaptic and RNA-processing proteomes. Two clusters of patients were discernible, but clustering occurred independent of mutation type. Together, these results demonstrate unique and common disease mechanisms between patients. Finally, in chapter 5 we introduced STXBP1 haploinsufficiency in neurons from multiple healthy donors and repaired a mutation in one patient line. We found that network activity patterns were affected by STXBP1 haploinsufficiency but to different extents between different donor cell lines. Moreover, we showed that the phenotypes change over time, also to different extents between different donor cell lines. Phenotype severity correlated with the reduction of MUNC18-1 expression levels, and baseline expression levels differed between cell lines. These results reveal cell line-specific characteristics of STXBP1 haploinsufficiency which may contribute to understanding the symptomatic heterogeneity of patients.