Reduced MUNC18-1 Levels, Synaptic Proteome Changes, and Altered Network Activity in STXBP1-Related Disorder Patient Neurons
Annemiek A. van Berkel, Hanna Charlotte Andrea Lammertse, Miriam Öttl, Frank Koopmans, Mala Misra‐Isrie, Marieke Meijer, Robertino Dilena, Peter M. van Hasselt, Marc Engelen, Mieke M. van Haelst, August B. Smit, Sophie van der Sluis, Ruud F. Toonen, Matthijs Verhage
Abstract
STXBP1-related disorder (STXBP1-RD) is a neurodevelopmental disorder caused by pathogenic variants in the STXBP1 gene. Its gene product MUNC18-1 organizes synaptic vesicle exocytosis and is essential for synaptic transmission. Patients present with developmental delay, intellectual disability and/or epileptic seizures, with high clinical heterogeneity. To date, the cellular deficits of STXBP1-RD patient neurons are unknown. Here, we combined live-cell imaging, electrophysiology, confocal microscopy and mass spec proteomics to characterize cellular phenotypes of iPSC-derived neurons from six STXBP1-RD patients, capturing shared features as well as phenotypic diversity among patients. Neurons from all patients showed normal in vitro development, morphology and synapse formation, but reduced MUNC18-1 RNA and protein levels. In addition, a proteome-wide screen identified dysregulation of proteins related to synapse function and RNA processes. Neuronal networks showed shared as well as patient-specific phenotypes in activity frequency, network irregularity and synchronicity, especially when networks were challenged by increasing excitability. No shared effects were observed in synapse physiology of single neurons, except a few patient-specific phenotypes. Similarities between functional and proteome phenotypes suggested two patient clusters, not explained by gene variant type. Together, these data show that decreased MUNC18-1 levels, dysregulation of synaptic proteins and altered network activity are shared cellular phenotypes of STXBP1-RD. The two patient clusters suggest distinctive pathobiology among subgroups of patients, providing a plausible explanation for the clinical heterogeneity. This phenotypic spectrum provides a framework for future validation studies and therapy design for STXBP1-RD.