Human Pluripotent Stem Cell-derived Models of STXBP1-associated Epilepsy.
Abstract number :
1.447
Submission category :
1. Basic Mechanisms / 1E. Models
Year :
2022
Submission ID :
2232876
Source :
www.aesnet.org
Presentation date :
12/3/2022 12:00:00 PM
Published date :
Nov 22, 2022, 05:28 AM
Authors :
Caroline Pearson, PhD – Weill Cornell Medicine; Tuo Ji, PhD – University of Michigan; Sandra Mojica-Perez, PhD – University of Michigan; Wei Nui, PhD – University of Michigan; Jack Parent, MD, PhD – University of Michigan; Whitney Parker, MD, PhD – Weill Cornell Medicine; M. Elizabeth Ross, MD, PhD – Brain and Mind Research Institute – Weill Cornell Medicine; Andrew Tidball, PhD – University of Michigan; Michael Uhler, PhD – University of Michigan; Chengbing Wang, PhD – Research Associate, Brain and Mind Research Institute, Weill Cornell Medicine
This is a Late Breaking abstract
Rationale: The Epilepsy Multiplatform Variant Prediction (EpiMVP) Center without walls is a highly integrated, multicenter collaboration aiming to develop prediction tools (EpiPred) for variants of uncertain significance (VUS) in epilepsy genes. We integrate machine learning, in silico modeling and biological data from in vitro and in vivo platforms to develop precise algorithms capable of classifying VUS with clinical impact as benign or pathogenic. Our initial efforts focus on non-ion channel/receptor epilepsy associated genes, including a critical component of the SNARE complex, Syntaxin binding protein 1 (STXBP1).
Methods: We have developed patient-derived induced pluripotent stem cells (iPSCs) and human embryonic stem cell (hESC) knockout (KO) lines to establish STXBP1 loss-of-function phenotypes. Using STXBP1 patient derived and healthy control iPSCs we have generated iNeurons through the forced expression of NGN2. Neuronal properties have been assessed using immunohistochemistry and Multi Electrode Array analyses.
Results: Using immunohistochemistry (IHC), we have shown that neurons with a patient specific STXBP1 mutation exhibit increased puncta size of STXBP1 protein, indicating increased aggregation. This aligns with biochemical analyses demonstrating decreased solubility of STXBP1 protein with the same mutation. Patient derived iNeurons also exhibit significantly increased cell death, and reduction in synapse formation compared to controls. Multi-electrode array (MEA) and calcium imaging analyses demonstrate that STXBP1 patient-derived neuron cultures are significantly less active than controls and little synchronous activity is established. Using patient-derived iPSCs and genetically engineered STXBP1 homozygous KO hESCs we have generated cortical organoids. IHC analyses of these 3D models demonstrated significantly increased neuronal cell death in both the patient derived and STXBP1 KO organoids at 90 days in vitro.
Conclusions: Thus, in 2D and 3D culture systems, in patient derived iPSCs and STXBP1 homozygous KO hESCs, STXBP1 mutant neurons exhibit increased cell death, reduced activity, and less synchronous firing. We are currently developing AAVs expressing STXBP1 VUS to test their ability to rescue these phenotypes in both 2D and 3D culture systems.
Funding: NIH NINDS U54 NS117170-01
Basic Mechanisms