Unravelling the role of the STX1B gene in genetic epilepsy syndromes using human induced pluripotent stem cells

Abstract

Synaptic vesicle release is a highly coordinated process that forms the basis for fast and efficient neuronal communication. The complex interplay of several presynaptic proteins fine-tunes the orchestration of the vesicle release machinery. Consequently, dysfunction of any single protein within this reaction chain can have detrimental consequences for synaptic transmission, ultimately disrupting the brain’s excitation/inhibition balance and manifesting as the clinical symptom of an epileptic seizure. Pathogenic variants in STX1B, encoding the presynaptic SNARE protein syntaxin-1B, have been associated with various epilepsy syndromes. To better understand the underlying pathophysiological mechanisms, the effects of STX1B variants have been investigated in different animal models. However, previous studies are constrained by the absence of phenotypic penetrance in the heterozygous state that reflects the patient’s condition, limiting their ability to accurately model the associated human neurological disorder. To more closely mimic the patient scenario, this thesis investigated the effect of STX1B variants in a human model system using induced pluripotent stem cells (iPSCs). For the generation of patient-derived cell lines, skin fibroblasts from individuals carrying pathogenic variants in the STX1B gene (G226R and InDel) were reprogrammed into iPSCs. An additional variant of interest (V216E) was inserted into a healthy control line by CRISPR/Cas9 gene editing. Leveraging the fast NGN2-based conversion of iPSCs into neurons, the variant-induced synaptic dysfunctions were electrophysiologically investigated at both network as well as single-cell level, with the latter allowing in-depth analysis of synapse function. While the three variants under investigation exhibit distinct synaptic dysfunctions of varying severity at the single-cell level, they eventually converge on a shared network phenotype, which is manifested by an increased burst and spike rate. Morphological and transcriptomic analyses point towards the implication of secondary mechanisms, initially triggered by the primary synaptic dysfunction, to be causative for a hyperexcitable network state. In addition, the large InDel variant negatively affects the syntaxin-1B protein levels, possibly due to protein instability. While the precise mechanisms linking primary synaptic dysfunction to the altered network state remain to be elucidated, this study provides novel interesting insights into the pathophysiology of STX1B-related synaptopathies, thereby paving the way for future complementary studies in more complex model systems.

Die Dissertation ist gesperrt bis zum 08. Oktober 2027 !