Variant-specific changes in persistent or resurgent Na+ current in SCN8A-EIEE13 iPSC-derived neurons

Mapping Intimacies ◽

10.1101/2020.01.16.909192 ◽

2020 ◽

Author(s):

Andrew M. Tidball ◽

Luis F. Lopez-Santiago ◽

Yukun Yuan ◽

Trevor W. Glenn ◽

Joshua L. Margolis ◽

...

Keyword(s):

Cortical Neurons ◽

Disease Modeling ◽

Wide Spectrum ◽

Sodium Current ◽

Specific Model ◽

Epileptic Encephalopathy ◽

Network Activity ◽

Patient Specific ◽

Unexpected Death ◽

Missense Variants

AbstractMissense variants in the voltage-gated sodium channel (VGSC) gene, SCN8A, are linked to early-infantile epileptic encephalopathy type 13 (EIEE13). EIEE13 patients exhibit a wide spectrum of intractable seizure types, severe developmental delay, movement disorders, and elevated risk of sudden unexpected death in epilepsy (SUDEP). The mechanisms by which SCN8A variants lead to epilepsy are poorly understood, although heterologous expression systems and mouse models have demonstrated altered sodium current (INa) properties. To investigate these mechanisms using a patient-specific model system, we generated induced pluripotent stem cells (iPSCs) from three patients with missense variants in SCN8A: p.R1872>L (P1); p.V1592>L (P2); and p.N1759>S (P3). Using small molecule differentiation into excitatory neurons, iPSC-derived neurons from all three patients displayed altered INa. P1 and P2 had elevated persistent INa, while P3 had increased resurgent INa compared to controls. Further analyses focused on one of the patients with increased persistent INa (P1) and the patient with increased resurgent INa (P3). Excitatory cortical neurons from both patients had prolonged action potential (AP) repolarization and shorter axon initial segment lengths compared to controls, the latter analyzed by immunostaining for ankyrin-G. Using doxycycline-inducible expression of the neuronal transcription factors Neurogenin 1 and 2 to synchronize differentiation of induced excitatory cortical-like neurons (iNeurons), we investigated network activity and response to pharmacotherapies. Both patient neurons and iNeurons displayed similar abnormalities in AP repolarization. Patient iNeurons showed increased burstiness that was sensitive to phenytoin, currently a standard treatment for EIEE patients, or riluzole, an FDA-approved drug used in amyotrophic lateral sclerosis and known to block persistent and resurgent INa, at pharmacologically relevant concentrations. Patch-clamp recordings showed that riluzole suppressed spontaneous firing and increased the AP firing threshold of patient-derived neurons to more depolarized potentials. Our results indicate that patient-specific neurons are useful for modeling EIEE13 and demonstrate SCN8A variant-specific mechanisms. Moreover, these findings suggest that patient-specific iPSC neuronal disease modeling offers a useful platform for discovering precision epilepsy therapies.

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Variant-specific changes in persistent or resurgent sodium current in SCN8A-related epilepsy patient-derived neurons

Brain ◽

10.1093/brain/awaa247 ◽

2020 ◽

Vol 143 (10) ◽

pp. 3025-3040 ◽

Cited By ~ 2

Author(s):

Andrew M Tidball ◽

Luis F Lopez-Santiago ◽

Yukun Yuan ◽

Trevor W Glenn ◽

Joshua L Margolis ◽

...

Keyword(s):

Action Potential ◽

Small Molecule ◽

Seizure Frequency ◽

Sodium Current ◽

Patient Specific ◽

Sodium Currents ◽

Missense Variants ◽

Induced Pluripotent ◽

Resurgent Current ◽

Action Potential Repolarization

Abstract Missense variants in the SCN8A voltage-gated sodium channel gene are linked to early-infantile epileptic encephalopathy type 13, also known as SCN8A-related epilepsy. These patients exhibit a wide spectrum of intractable seizure types, severe developmental delay, movement disorders, and elevated risk of sudden unexpected death in epilepsy. The mechanisms by which SCN8A variants lead to epilepsy are poorly understood, although heterologous expression systems and mouse models have demonstrated altered sodium current properties. To investigate these mechanisms using a patient-specific model, we generated induced pluripotent stem cells from three patients with missense variants in SCN8A: p.R1872>L (Patient 1); p.V1592>L (Patient 2); and p.N1759>S (Patient 3). Using small molecule differentiation into excitatory neurons, induced pluripotent stem cell-derived neurons from all three patients displayed altered sodium currents. Patients 1 and 2 had elevated persistent current, while Patient 3 had increased resurgent current compared to controls. Neurons from all three patients displayed shorter axon initial segment lengths compared to controls. Further analyses focused on one of the patients with increased persistent sodium current (Patient 1) and the patient with increased resurgent current (Patient 3). Excitatory cortical neurons from both patients had prolonged action potential repolarization. Using doxycycline-inducible expression of the neuronal transcription factors neurogenin 1 and 2 to synchronize differentiation of induced excitatory cortical-like neurons, we investigated network activity and response to pharmacotherapies. Both small molecule differentiated and induced patient neurons displayed similar abnormalities in action potential repolarization. Patient induced neurons showed increased burstiness that was sensitive to phenytoin, currently a standard treatment for SCN8A-related epilepsy patients, or riluzole, an FDA-approved drug used in amyotrophic lateral sclerosis and known to block persistent and resurgent sodium currents, at pharmacologically relevant concentrations. Patch-clamp recordings showed that riluzole suppressed spontaneous firing and increased the action potential firing threshold of patient-derived neurons to more depolarized potentials. Two of the patients in this study were prescribed riluzole off-label. Patient 1 had a 50% reduction in seizure frequency. Patient 3 experienced an immediate and dramatic seizure reduction with months of seizure freedom. An additional patient with a SCN8A variant in domain IV of Nav1.6 (p.V1757>I) had a dramatic reduction in seizure frequency for several months after starting riluzole treatment, but then seizures recurred. Our results indicate that patient-specific neurons are useful for modelling SCN8A-related epilepsy and demonstrate SCN8A variant-specific mechanisms. Moreover, these findings suggest that patient-specific neuronal disease modelling offers a useful platform for discovering precision epilepsy therapies.

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Pathogenic MAST3 variants in the STK domain are associated with epilepsy

10.1101/2021.03.09.434675 ◽

2021 ◽

Author(s):

Egidio Spinelli ◽

Kyle R Christensen ◽

Emily Bryant ◽

Amy Schneider ◽

Jennifer Rakotomamonjy ◽

...

Keyword(s):

De Novo ◽

Neurodevelopmental Disorder ◽

Expression Patterns ◽

Prenatal Development ◽

Epileptic Encephalopathy ◽

Patient Specific ◽

Gain Of Function ◽

Missense Variants ◽

Excitatory Neurons

AbstractObjectiveThe MAST family of microtubule-associated serine-threonine kinases (STK) have distinct expression patterns in the developing and mature human and mouse brain. To date, only MAST1 has been associated with neurological disease, with de novo variants in individuals with a neurodevelopmental disorder, including a mega corpus callosum.MethodsUsing exome sequencing we identify MAST3 missense variants in individuals with epilepsy. We also assess the effect of these variants on the ability of MAST3 to phosphorylate the target gene product ARPP-16 in HEK293T cells.ResultsWe identify de novo missense variants in the STK domain in 11 individuals, including two recurrent variants p.G510S (n=5) and p.G515S (n=3). All 11 individuals had Developmental and epileptic encephalopathy, with 8 having normal development prior to seizure onset at < 2 years of age. All patients developed multiple seizures types, while 9/11 had seizures triggered by fever and 9/11 had drug-resistant seizures. In vitro analysis of HEK293T cells transfected with MAST3 cDNA carrying a subset of these patient-specific missense variants demonstrated variable but generally lower expression, with concomitant increased phosphorylation of the MAST3 target, ARPP-16, compared to wildtype. These findings suggest the patient-specific variants may confer MAST3 gain-of-function. Moreover, single-nuclei RNA sequencing and immunohistochemistry shows that MAST3 expression is restricted to excitatory neurons in the cortex late in prenatal development and postnatally.InterpretationIn summary, we describe MAST3 as a novel epilepsy-associated gene with a potential gain-of-function pathogenic mechanism that may be primarily restricted to excitatory neurons in the cortex.

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Faculty Opinions recommendation of Missense variants in the N-terminal domain of the A isoform of FHF2/FGF13 cause an X-linked developmental and epileptic encephalopathy.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.739117931.793580692 ◽

2020 ◽

Author(s):

Elizabeth Palmer

Keyword(s):

Epileptic Encephalopathy ◽

Missense Variants ◽

Terminal Domain

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Altered network properties in C9ORF72 repeat expansion cortical neurons are due to synaptic dysfunction

Molecular Neurodegeneration ◽

10.1186/s13024-021-00433-8 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Emma M. Perkins ◽

Karen Burr ◽

Poulomi Banerjee ◽

Arpan R. Mehta ◽

Owen Dando ◽

...

Keyword(s):

Synaptic Plasticity ◽

Synaptic Vesicle ◽

Cortical Neurons ◽

Electrode Array ◽

Repeat Expansion ◽

Cortical Network ◽

Synaptic Function ◽

Network Activity ◽

Cortical Function ◽

Network Function

Abstract Background Physiological disturbances in cortical network excitability and plasticity are established and widespread in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients, including those harbouring the C9ORF72 repeat expansion (C9ORF72RE) mutation – the most common genetic impairment causal to ALS and FTD. Noting that perturbations in cortical function are evidenced pre-symptomatically, and that the cortex is associated with widespread pathology, cortical dysfunction is thought to be an early driver of neurodegenerative disease progression. However, our understanding of how altered network function manifests at the cellular and molecular level is not clear. Methods To address this we have generated cortical neurons from patient-derived iPSCs harbouring C9ORF72RE mutations, as well as from their isogenic expansion-corrected controls. We have established a model of network activity in these neurons using multi-electrode array electrophysiology. We have then mechanistically examined the physiological processes underpinning network dysfunction using a combination of patch-clamp electrophysiology, immunocytochemistry, pharmacology and transcriptomic profiling. Results We find that C9ORF72RE causes elevated network burst activity, associated with enhanced synaptic input, yet lower burst duration, attributable to impaired pre-synaptic vesicle dynamics. We also show that the C9ORF72RE is associated with impaired synaptic plasticity. Moreover, RNA-seq analysis revealed dysregulated molecular pathways impacting on synaptic function. All molecular, cellular and network deficits are rescued by CRISPR/Cas9 correction of C9ORF72RE. Our study provides a mechanistic view of the early dysregulated processes that underpin cortical network dysfunction in ALS-FTD. Conclusion These findings suggest synaptic pathophysiology is widespread in ALS-FTD and has an early and fundamental role in driving altered network function that is thought to contribute to neurodegenerative processes in these patients. The overall importance is the identification of previously unidentified defects in pre and postsynaptic compartments affecting synaptic plasticity, synaptic vesicle stores, and network propagation, which directly impact upon cortical function.

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Prediction of 3D Cardiovascular hemodynamics before and after coronary artery bypass surgery via deep learning

Communications Biology ◽

10.1038/s42003-020-01638-1 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Gaoyang Li ◽

Haoran Wang ◽

Mingzi Zhang ◽

Simon Tupin ◽

Aike Qiao ◽

...

Keyword(s):

Deep Learning ◽

Artery Bypass ◽

Computational Cost ◽

Specific Model ◽

Arterial System ◽

Patient Specific ◽

Cardiovascular Hemodynamics ◽

Before And After ◽

Deep Learning Network ◽

Cfd Method

AbstractThe clinical treatment planning of coronary heart disease requires hemodynamic parameters to provide proper guidance. Computational fluid dynamics (CFD) is gradually used in the simulation of cardiovascular hemodynamics. However, for the patient-specific model, the complex operation and high computational cost of CFD hinder its clinical application. To deal with these problems, we develop cardiovascular hemodynamic point datasets and a dual sampling channel deep learning network, which can analyze and reproduce the relationship between the cardiovascular geometry and internal hemodynamics. The statistical analysis shows that the hemodynamic prediction results of deep learning are in agreement with the conventional CFD method, but the calculation time is reduced 600-fold. In terms of over 2 million nodes, prediction accuracy of around 90%, computational efficiency to predict cardiovascular hemodynamics within 1 second, and universality for evaluating complex arterial system, our deep learning method can meet the needs of most situations.

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Depression patient-derived cortical neurons reveal potential biomarkers for antidepressant response

Translational Psychiatry ◽

10.1038/s41398-021-01319-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yishai Avior ◽

Shiri Ron ◽

Dana Kroitorou ◽

Claudia Albeldas ◽

Vitaly Lerner ◽

...

Keyword(s):

Cortical Neurons ◽

Antidepressant Treatment ◽

Patient Specific ◽

Specific Gene ◽

Specific Information ◽

Patient Response ◽

Patient Will ◽

Gene Expression Alterations ◽

Treatment Alternatives

AbstractMajor depressive disorder is highly prevalent worldwide and has been affecting an increasing number of people each year. Current first line antidepressants show merely 37% remission, and physicians are forced to use a trial-and-error approach when choosing a single antidepressant out of dozens of available medications. We sought to identify a method of testing that would provide patient-specific information on whether a patient will respond to a medication using in vitro modeling. Patient-derived lymphoblastoid cell lines from the Sequenced Treatment Alternatives to Relieve Depression study were used to rapidly generate cortical neurons and screen them for bupropion effects, for which the donor patients showed remission or non-remission. We provide evidence for biomarkers specific for bupropion response, including synaptic connectivity and morphology changes as well as specific gene expression alterations. These biomarkers support the concept of personalized antidepressant treatment based on in vitro platforms and could be utilized as predictors to patient response in the clinic.

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Evaluating Design of Abdominal Aortic Aneurysm Endografts in a Patient-Specific Model Using Computational Fluid Dynamics

Journal of Medical Devices ◽

10.1115/1.3589227 ◽

2011 ◽

Vol 5 (2) ◽

Cited By ~ 2

Author(s):

Polina A. Segalova ◽

Guanglei Xiong ◽

K. T. Rao ◽

Christopher K. Zarins ◽

Charles A. Taylor

Keyword(s):

Fluid Dynamics ◽

Computational Fluid Dynamics ◽

Abdominal Aortic Aneurysm ◽

Aortic Aneurysm ◽

Specific Model ◽

Patient Specific ◽

Abdominal Aortic

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Matrigel Mattress

Circulation Research ◽

10.1161/circresaha.115.307580 ◽

2015 ◽

Vol 117 (12) ◽

pp. 995-1000 ◽

Cited By ~ 83

Author(s):

Tromondae K. Feaster ◽

Adrian G. Cadar ◽

Lili Wang ◽

Charles H. Williams ◽

Young Wook Chun ◽

...

Keyword(s):

Drug Discovery ◽

Single Cell ◽

Troponin I ◽

Disease Modeling ◽

Sodium Current ◽

Culture Method ◽

Adult Rabbit ◽

Standard Equipment ◽

Cell Contractility ◽

Contractile Performance

Rationale: The lack of measurable single-cell contractility of human-induced pluripotent stem cell–derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery. Objective: To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs. Methods and Results: Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (<0.1-mm thick 1:60 diluted Matrigel, control hiPSC-CMs). Compared with control hiPSC-CMs, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection were comparable with those of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CMs were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CMs, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents, such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity because of a 2-fold increase in sodium current ( I Na ). Conclusions: The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing.

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