Morphological and electrophysiological characterization of STXBP1 patient iPSC-derived cortical-like pyramidal neurons
Abstract number :
3.391
Submission category :
1. Basic Mechanisms / 1B. Epileptogenesis of genetic epilepsies
Year :
2021
Submission ID :
1886447
Source :
www.aesnet.org
Presentation date :
12/6/2021 12:00:00 PM
Published date :
Nov 22, 2021, 06:56 AM
Authors :
Yukun Yuan, PhD - University of Michigan; Tuo Ji, Ph.D. - Department of Neurology - University of Michigan; Caroline Pearson, Ph.D. - Departments of Neurology and Neuroscience, Brain and Mind Research Institute - Weill Cornell Medical College; Whitney Parker, M.D., Ph.D. - Departments of Neurology and Neuroscience, Brain and Mind Research Institute - Weill Cornell Medical College; Michael Uhler, Ph.D. - Professor, Department of Biochemistry, University of Michigan; Luis Lopez-Santiago, Ph.D. - Department of Pharmacology - University of Michigan; Margaret E Ross, M.D., Ph.D. - Professor, Chair, Departments of Neurology and Neuroscience, Brain and Mind Research Institute, Weill Cornell Medical College; Jack Parent, M.D. - Professor, Department of Neurology, University of Michigan; Lori Isom, Ph.D. - Professor, Chair, Department of Pharmacology, Department of Neurology, University of Michigan
Rationale: STXBP1, encoding the presynaptic protein Syntaxin-binding protein 1, also called Munc18-1, is essential for synaptic vesicle fusion and neurotransmitter release. Variants in STXBP1 are associated with a spectrum of developmental and epileptic encephalopathies (DEEs), however, the underlying cellular and molecular pathophysiological mechanisms remain poorly understood. Here, as the first step, we characterized changes in morphology, synaptic transmission, and neuronal excitability of human induced pluripotent stem cell (iPSC)-derived cortical neurons from control and STXBP1 DEE patients.
Methods: Fibroblasts from an individual with STXBP1 DEE due to a heterozygous p.G544Vfs*2 variant were obtained and reprogrammed into iPSCs by Sendai viral transduction of OCT3/4, SOX2, KLF-4, and L-MYC. Cortical-like excitatory neurons were generated from control and patient iPSCs by doxycycline (dox)-inducible expression of human neurogenin-2 (NGN2) using PiggyBac vectors. Tuj1 positive neurons were examined for localization of pre- and post-synaptic markers, VGlut1 and PSD95, respectively. Spontaneous excitatory postsynaptic currents (sEPSCs), action potentials, and firing pattern of iPSC-derived iNeurons at 28-32 days after the initiation of 7-day dox treatment were recorded using whole cell patch-clamp techniques.
Results: STXBP1 patient neurons displayed reduced overlap of VGlut1 and PSD95 labeling, suggesting fewer functional synapses compared to control neurites. Whole cell patch-clamp recordings showed significant reductions in both sEPSC frequency (0.33 ± 0.07 Hz, n=15) and amplitudes (8.84 ± 1.26 pA, n=15) in STXBP1 patient neurons compared to controls (4.49 ± 0.88 Hz and 30.54 ± 3.66 pA, respectively, n=13, p< 0.001), suggesting that spontaneous neurotransmitter release from presynaptic terminals is significantly impaired. Consistent with this result, the patient neurons had significantly higher threshold potentials for action potential initiation, higher sensitivity to depolarization-induced block, slower maximum rise rates, and lower firing frequency (P< 0.001, n=29) compared to controls (n=21). In contrast, there were no significant differences in resting membrane potential or peak amplitude of the action potential between genotypes. Interestingly, a majority of patient neurons showed increased sag current at hyperpolarized current injections, suggesting an increase in hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel-mediated currents.
Basic Mechanisms