Reduced MUNC18-1 Levels, Synaptic Proteome Changes, and Altered Network Activity in STXBP1-Related Disorder Patient Neurons
van Berkel, Annemiek Arienne; Lammertse, Hanna Charlotte Andrea; Öttl, Miriam; Koopmans, Frank; Misra-Isrie, Mala; Meijer, Marieke; Dilena, Robertino; van Hasselt, Peter Marin; Engelen, Marc; van Haelst, Mieke; Smit, August Benjamin; van der Sluis, Sophie; Toonen, Ruud Franciscus; Verhage, Matthijs
(2024) Biological psychiatry global open science, volume 4, issue 1, pp. 284 - 298
(Article)
Abstract
Background: 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
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neurons of patients with STXBP1-RD are unknown. Methods: We combined live-cell imaging, electrophysiology, confocal microscopy, and mass spectrometry proteomics to characterize cellular phenotypes of induced pluripotent stem cell–derived neurons from 6 patients with STXBP1-RD, capturing shared features as well as phenotypic diversity among patients. Results: 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 for a few patient-specific phenotypes. Similarities between functional and proteome phenotypes suggested 2 patient clusters, not explained by gene variant type. Conclusions: 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 2 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.
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Keywords: iPSC, MUNC18-1, Neurodevelopmental disorder, SNAREopathy, STXBP1-related disorder, Synaptic transmission, Phychiatric Mental Health, Clinical Neurology, Psychiatry and Mental health, Biological Psychiatry
ISSN: 2667-1743
Publisher: Elsevier BV
Note: Funding Information: This work was supported by a Pilot Grant from the Orphan Disease Center/STXBP1 Foundation (Grant No. MDBR-20-136-STXBP1 [to MV]), Netherlands Scientific Organisation Gravitation program BRAINSCAPES (Grant No. NWO 024.004.012 [to MV and SvdS]), COSYN Horizon 2020 Program of the European Union (Grant No. 667301 [to MV]), Lundbeck Foundation (Grant No. R277-2018-802 [to MV]), ZonMw (Veni Grant No. 09150161810052 [to MM]), and Netherlands Scientific Organisation and De Hersenstichting under the frame of NEURON Cofund ERA-NET SNAREopathy (Grant No. 013-17-002 [to RFT]). AAvB, HCAL, RFT, and MV conceptualized the project and designed experiments. RD provided fibroblasts of the K308X patient. PMvH provided fibroblasts and clinical assessments of the D262V patient. MM-I, ME, and MvH performed clinical assessments on the D207G, S241fs, R235X, and intronic patients. AAvB and HCAL acquired and analyzed immunocytochemical, Western blotting, and RNA data. HCAL and MÖ acquired electrophysiological data, and HCAL analyzed the data, supervised by MM. AAvB acquired and analyzed calcium imaging data. AAvB and FK analyzed proteomics data under the supervision of ABS. SvdS advised on and supervised the statistical analyses. AAvB, HCAL, RFT, and MV wrote the manuscript with input from all authors. We thank the STXBP1-RD patients and their families for participating in our research. We thank Desiree Schut, Lisa Laan, and Eline Kompanje for assistance in cell culture, Joost Hoetjes for assistance in RNA isolation and quantitative polymerase chain reaction, and Iryna Paliukhovich and Ka Wan Li for expert help with the mass spectrometry experiments. We thank Vincent Huson for developing scripts used for analysis of the electrophysiological data and Josefin Werme for assistance in the analysis of copy number variants from single nucleotide polymorphism arrays for quality control purposes. We thank Vivi Heine for providing iPSC lines from control individuals. We thank Jessica Bos for processing medical records of the patients, Nathalie Bogaards for performing skin biopsies, and Hanne Meijers-Heijboer for clinical assistance. MV and RFT are founders of Neurospector, which offers contract research services for STXBP1-RD using cellular models and methodology similar to those described in this study. All other authors report no biomedical financial interests or potential conflicts of interest. Funding Information: This work was supported by a Pilot Grant from the Orphan Disease Center / STXBP1 Foundation (Grant No. MDBR-20-136-STXBP1 [to MV]), Netherlands Scientific Organisation Gravitation program BRAINSCAPES (Grant No. NWO 024.004.012 [to MV and SvdS]), COSYN Horizon 2020 Program of the European Union (Grant No. 667301 [to MV]), Lundbeck Foundation (Grant No. R277-2018-802 [to MV]), ZonMw (Veni Grant No. 09150161810052 [to MM]), and Netherlands Scientific Organisation and De Hersenstichting under the frame of NEURON Cofund ERA-NET SNAREopathy (Grant No. 013-17-002 [to RFT]). Publisher Copyright: © 2023 The Authors
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