Modeling STXBP1 Loss of Function Using Human Pluripotent Stem Cell Derived 2D and 3D Models
Abstract number :
1.016
Submission category :
1. Basic Mechanisms / 1B. Epileptogenesis of genetic epilepsies
Year :
2022
Submission ID :
2204530
Source :
www.aesnet.org
Presentation date :
12/3/2022 12:00:00 PM
Published date :
Nov 22, 2022, 05:25 AM
Authors :
Kyle Stokes, Ph. D. – University of Michigan; Wei Niu, PhD – Research Investigator, Neurology, University of Michigan; Yukun Yuan, PhD – Research Assistant Professor, Pharmacology, University of Michigan; Tuo Ji, PhD – Research Specialist, Neurology, University of Michigan; Sandra Mojica-Perez, PhD – Research Lab Sepcialist Senior, Neurology, University of Michigan; Andrew Tidball, PhD – Research Investigator, Neurology, University of Michigan; Caroline Pearson, PhD – Assistant Professor, Neuroscience, Weill Cornel Medical College; Whitney Parker, MD, PhD – Resident, Neuroscience, Weill Cornel Medical College; Chengbing Wang, PhD – enior Research Associate, Neuroscience, Weill Cornel Medical College; Margaret Ross, MD, PhD – Professor, Neurogenetics and Development, Weill Cornel Medical College; Lori Isom, PhD – Professor, Pharmacology, University of Michigan; Michael Uhler, PhD – Professor, Neurology, University of Michigan; Jack Parent, MD, PhD – Professor, Neurology, University of Michigan
This abstract has been invited to present during the Basic Science Poster Highlights poster session
Rationale: The Epilepsy Multiplatform Variant Prediction (EpiMVP) Center Without Walls is a collaboration between 6 institutions to develop an epilepsy prediction (EpiPred) software that predicts the pathogenicity of genetic variants of uncertain significance (VUS) in epilepsy associated genes. One gene of interest, Syntaxin binding protein 1 (STXBP1), regulates vesicular membrane fusion and release from the presynaptic neuron. Loss of function (LoF) variants in the STXBP1 gene are associated with severe early onset epileptic encephalopathies. Although loss of STXBP1 has been shown to reduce excitatory neurotransmitter release, it is unclear how this deficit impairs neuronal activity and results in epilepsy. In this study, STXBP1 patient derived and CRISPR/Cas9 gene edited human pluripotent stem cells (hPSCs) were used to generate human cortical organoids (hCOs), a 3D cell culture technique, and Neurogenin-2 (NGN2)-induced 2D cortical-like pyramidal neurons (iNeurons) to investigate how STXBP1 LoF leads to altered neurodevelopment and epilepsy.
Methods: We reprogrammed patient fibroblasts using the Yamanaka factors (OCT3/4, SOX2, KLF4, and L-MYC) to generate induced PSCs (iPSCs). Heterozygous and homozygous STXBP1 LoF mutations were generated in other hPSC lines using CRISPR/Cas9 genome editing and were compared to isogenic controls. We then differentiated these hPSC lines into hCOs or doxycycline-inducible NGN2 iNeurons. The organoid protocol used is a novel single rosette organoid protocol that was developed in the Parent lab. The organoids were collected at multiple timepoints and subjected to histological analyses. Later-stage hCOs were assessed for network activity on multielectrode array (MEA) plates. Action potentials were recorded from organoid slices and iNeurons using whole cell patch-clamping.
Results: Both patient iPSC-derived hCOs and CRISPR homozygous knockout (KO) hCOs display increased cell death as indicated by cleaved Caspase 3 immunolabeling at day 90, and a reduction in cortical-like excitatory neurons marked by CTIP2 and SATB2 when compared to isogenic controls. MEA recordings of STXBP1 KO hCOs at 6-7 months showed reduced mean firing rate compared to isogenic controls. In addition, organoid slice patch-clamp recordings showed significant reduction of action potentials in STXBP1 heterozygous KO hCOs compared to isogenic controls which is consistent with electrophysiological findings in STXBP1 patient iPSC-derived iNeurons.
Conclusions: We established epilepsy-like phenotypes associated with STXBP1 LoF in 2D and 3D cell culture models. Our results suggest that STXBP1 LoF disrupts cortical development and impairs spontaneous neurotransmitter release from presynaptic terminals. Future experiments will involve the forced expression of STXBP1 wildtype, pathogenic, benign and VUS in STXBP1 KO hPSCs to assay for rescue of the observed LoF phenotypes. These results will be fed into EpiPred to improve its predictive power for epilepsy related VUS. These data and the enhanced predictive power of EpiPred may be a beneficial resource for the diagnosis and treatment of epilepsy patients.
Funding: Supported by NIH (NINDS) U54NS117170
Basic Mechanisms