The Price of Prominence: STXBP1/MUNC18-1 in brain (dys)function

Neuronal functioning and viability strongly depend on presynaptic protein MUNC18-1. In absence of MUNC18-1, mouse neurons die, which is independent from its known function in synaptic transmission. Pathogenic variants in its gene, STXBP1, are among the most common associated with developmental delay and epilepsy. The general aim of this thesis was to gain more insight in why MUNC18-1 is such an essential protein in neurons beyond synaptic transmission, and how pathogenic variants lead to disease. In Chapter 2, we investigated which cell death pathway(s) are involved in MUNC18-1 and syntaxin-1-dependent degeneration. We show that the last stages before cell death are characterized by gradual loss of neurites, which is not observed in other well-characterized forms of neuronal cell death. In the final stage, neurons express markers associated with apoptosis, yet inhibition of apoptosis is insufficient to halt cell death. We conclude that depletion of MUNC18-1 or syntaxin-1 leads to atypical cell death ultimately involving, but not driven by, apoptosis. In Chapter 3, we examined which biological pathways critically depend on MUNC18-1. We show that absence of MUNC18-1 results in extensive remodelling of the neuronal transcriptome and proteome. Dysregulated proteins are primarily involved in neuron development and synapse function. Many of the downregulated proteins are normally upregulated during early development. Thus, MUNC18-1 may act as a critical regulator of developmental and synaptic protein expression in immature neurons. In Chapter 4, we investigated whether loss of MUNC18-1 leads to defects in membrane transport routes, which may explain the observed abnormal Golgi morphology and degeneration in null neurons. We conclude that loss of MUNC18-1 leads to defects in retro- but not anterograde membrane trafficking pathways, which provides a plausible explanation for the abnormal Golgi morphology. In Chapter 5, we investigated the cellular phenotype of the first homozygous STXBP1 variant reported in patients. Neurons expressing L446F demonstrate a two-fold increase in evoked synaptic transmission. We conclude that the homozygous L446F variant leads to an opposing cellular phenotype than previously reported for heterozygous variants, yet results in comparable clinical symptoms. In Chapter 6, we developed a gene-tailored in silico classifier which predicts pathogenicity with high confidence, and outperforms currently available (generic) classifiers. Next, we examined which biological features are predictive for pathogenicity. We show in an allelic series of variants that protein instability, reduced MUNC18-1 expression levels and impaired function in SNARE fusion are shared features of pathogenic variants. The scores from the in silico classifier highly correlate to the experimental data. Thus, this extendable framework increased pathogenicity prediction of MUNC18-1 variants and improved understanding on underlying disease mechanisms. In Chapter 7, we evaluated commonly used designs involved iPSC-based disease models in terms of the research questions they address, statistical analysis and power considerations. We show that culture batch and interindividual variation contributes to overall experimental variation and should be taken into account in the statistical analysis. To foster future study designs, we deliver a web application to enable researchers to perform power analyses on their own data. In Chapter 8, we studied cellular phenotypes of iPSC-derived neurons from six STXBP1-Syndrome patients. We show that patient neurons present lower MUNC18-1 levels as well as dysregulation of many other proteins related to synapse function. Neuronal networks demonstrate altered frequency, network irregularity and synchronicity, whereas synapse physiology of single neurons is unaffected. We conclude that reduced MUNC18-1 levels, dysregulation of synaptic proteins and altered network activity are shared cellular phenotypes of STXBP1- Syndrome neurons. Taken together, we now better understand when variants in STXBP1 are disease-causing or neutral, and how these variants affect neuronal functioning