Biology subjects

Thompson, C. H.

Publications and source records attributed to Thompson, C. H..

12 recordsLinked to original sources

bioRxiv2026

ATP1A3 dysfunction alters excitability and developmental trajectory of human neurons in alternating hemiplegia of childhood

Pathogenic variants in ATP1A3, encoding the neuronal Na/K-ATPase, are associated with a spectrum of neuro-developmental disorders including alternating hemiplegia of childhood (AHC). ATP1A3 is expressed throughout the cortex during early development, and later becomes enriched in GABAergic neurons. Here, we interrogated the underlying mechanisms and neurophysiological consequences of a recurrent AHC-associated variant (ATP1A3-G947R) in human induced pluripotent stem cell (hiPSC) derived glutamatergic and GABAergic neu-rons. We found that glutamatergic and GABAergic AHC neurons exhibited smaller ouabain-sensitive pump cur-rents compared to isogenic mutation-corrected control neurons. This impairment of pump function correlated with a depolarized potassium reversal potential, lower intracellular potassium concentration, and depolarized resting membrane potential for both cell types compared to isogenic control neurons. Additionally, using a Na+ sensitive dye, we observed that AHC neurons had a higher basal intracellular Na+ concentration that recovered more slowly following Na+ loading compared to isogenic control neurons. As an additional consequence of impaired pump function and abnormal Na+ dynamics, glutamatergic and GABAergic AHC neurons were more prone to depolarization block. Finally, the time course by which GABA neurotransmission switches from excita-tory to inhibitory was delayed in glutamatergic AHC neurons compared to isogenic controls. Our findings pro-vide evidence that impairment of ATP1A3 function has direct effects on ionic homeostasis, leading to cascading effects on intrinsic neuronal firing and maturation of GABAergic neurotransmission that advance understanding of the pathophysiology of AHC.

neuroscience↗

bioRxiv2026

Elevated neuronal excitability and seizure susceptibility in a mouse model of SCN2A-related developmental and epileptic encephalopathy

Pathogenic variants in SCN2A cause a spectrum of neurodevelopmental disorders, including developmental and epileptic encephalopathies (DEE). The patient-associated SCN2A-p.E430A variant selectively shifts voltage-dependence of activation, a mechanism predicted to enhance neuronal excitability. To investigate the functional consequences of the SCN2A-E430A variant, we generated the novel Scn2aE430A mouse model and assessed neuronal excitability, brain activity, seizure susceptibility, and phenytoin responsiveness. Heterozygous Scn2aE430A mice retained normal Scn2a expression levels and did not exhibit premature lethality. Hippocampal pyramidal neurons from heterozygous Scn2aE430A mice were hyperexcitable compared to wild-type neurons. Enhanced long-term potentiation was observed in the CA1 circuit of Scn2aE430A mice. Video-EEG data revealed recurrent epileptiform discharges and spectral abnormalities, but no spontaneous generalized seizure events. In multiple seizure-induction assays, Scn2aE430A mice had no difference in latency to first seizure signs, but exhibited faster seizure generalization and more lethality compared to wild-type mice, indicating alterations in seizure propagation and/or cessation rather than seizure initiation. Pretreatment with phenytoin improved seizure outcomes across all seizure induction methods. Together, these findings demonstrate that selective disruption of Scn2a activation gating is sufficient to drive neuronal hyperexcitability and network dysfunction, establishing the Scn2aE430A mouse as clinically relevant and pharmacologically tractable model of SCN2A-related DEE.

neuroscience↗

bioRxiv2026

KCNH1 variants associated with neurodevelopmental disorders exhibit prominent gain-of-function

Pathogenic variants in KCNH1, encoding the human ether-a-go-go voltage-gated potassium channel (EAG1, KV10.1), are associated with the neurodevelopmental disorders Temple-Baraitser syndrome (TBS), Zimmermann-Laband syndrome (ZLS), and epileptic encephalopathy. Investigating the functional consequences of KCNH1 variants can provide information about molecular and cellular pathophysiological mechanisms, and help guide therapeutic directions. In this study, we determined the functional properties of 26 disease-associated KCNH1 variants including three recurrent and five that were previously unpublished. Findings were compared to the wildtype (WT) channel along with six rare population variants. Our approach used automated patch clamp recording of heterologously expressed KCNH1 variants in both the homozygous state (variant alone) and the clinically-relevant heterozygous state (variant co-expressed with WT channels). We also examined the impact of alternative splice isoforms on functional consequences of specific variants. We demonstrated that the majority of disease-associated KCNH1 variants exhibit features consistent with gain-of-function including hyperpolarized voltage-dependence of activation, accelerated activation kinetics, or both that promote hyperpolarization of the resting membrane potential. By contrast, one unique variant discovered previously in an autism cohort had a depolarized voltage-dependence of activation and was associated with a depolarized resting potential in cells. Pharmacological testing of KCNH1 variants demonstrated differences in potency for channel block by imipramine and an investigational non-selective potassium channel blocker (LY97241) for specific variants. Collectively, our findings support gain-of-function as the most common, albeit not exclusive, molecular mechanism underlying KCNH1-related disorders and suggest opportunities for potential therapeutic drug repurposing strategies for these conditions.

neuroscience↗

bioRxiv2025

Epilepsy-associated Variants of a Single SCN1A Codon exhibit Divergent Functional Properties

ObjectivePathogenic variants in SCN1A, which encodes the voltage gated sodium channel NaV1.1, are associated with multiple epilepsy syndromes exhibiting a range of clinical severity. Loss or gain of function SCN1A variants are reported in different syndromes including Dravet syndrome, which is associated with loss-of-function whereas neonatal/infantile-onset developmental and epileptic encephalopathy (DEE) is associated with gain-of-function. Strategies to predict SCN1A variant pathogenicity and dysfunction have been proposed but are limited by available training data. We investigated the functional properties of four epilepsy-associated SCN1A variants affecting the same codon and sought to correlate channel dysfunction with phenotype. MethodsWhole-cell manual patch-clamp recording was performed on heterologously-expressed NaV1.1 variants. Structural modeling of NaV1.1 variant proteins was conducted using AlphaFold 3. ResultsWe describe an individual with early infantile onset DEE associated with SCN1A-I1347T, and identified three additional cases from the literature or ClinVar with distinct variation of the same codon (I1347N, I1347V, I1347F). Functional studies demonstrated mixed gain and loss of function properties for I1347T, I1347V, and I1347F, but complete loss-of-function for I1347N. Structural models suggest important interactions between isoleucine-1347 and the sixth transmembrane helices of domains 3 and 4 that are disrupted most significantly with asparagine replacement at this position (I1347N). InterpretationPathogenic variants in SCN1A involving the same codon can produce divergent functional effects. Our findings suggest that predicting specific functional effects of SCN1A variants should not rely heavily on position in the protein.

neuroscience↗

bioRxiv2024

Optimizing clinical interpretability of functional evidence in epilepsy-related ion channel variants

Variants in genes encoding the voltage-gated ion channels are among the most common monogenic causes of epilepsy and neurodevelopmental disorders. Functional effects of a variant are increasingly important for diagnosis and therapeutic decisions. To incorporate knowledge regarding functional consequences in formal clinical variant interpretation, we developed an approach for evaluating multiple functional measurements within the Bayesian framework of the modified ACMG/AMP guidelines. We analyzed 216 functional assessments of 191 variants in SCN1A (n=74), SCN2A (n=66), SCN3A (n=18), and SCN8A (n=33). Of 20 commonly measured biophysical parameters, the most frequent drivers of overall functional consequence were persistent current (f=0.54), voltage dependence of activation (f=0.51), and voltage dependence of fast inactivation (f=0.40) for gain-of-function and peak current (f=0.87) for loss-of-function. By comparing measurements of 23 benign variants, we determined thresholds by which published data on these four parameters confer Strong evidence of variant pathogenicity (likelihood ratio > 18.7) under the ACMG/AMP rubric. Similarly, we delineated evidence weights for the most common epilepsy-related potassium channel gene, KCNQ2, through reports of 80 pathogenic and 24 benign variants, accounting for heterozygous and homozygous experimental conditions. We collected the resulting categorization of functional data into FENICS, a biomedical ontology of 152 standardized terms for coherent annotation of electrophysiological results. Across 271 variants in SCN1A/2A/3A/8A and KCNQ2, 1,731 annotations are available in ClinVar, facilitating use of this evidence in variant classification. In summary, we introduce and apply an ACMG/AMP-calibrated framework for electrophysiological studies in epilepsy-related channelopathies to delineate the impact of functional evidence on clinical variant interpretation.

genetics↗

bioRxiv2023

Molecular and Cellular Context Influences SCN8A Variant Function

Pathogenic variants in SCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, are associated with neurodevelopmental disorders including epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by the use of a neonatal-expressed alternatively spliced isoform of NaV1.6 (NaV1.6N), and engineered mutations to render the channel tetrodotoxin (TTX) resistant. In this study, we investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in two developmentally regulated splice isoforms (NaV1.6N, NaV1.6A). We employed automated patch clamp recording to enhance throughput, and developed a novel neuronal cell line (ND7/LoNav) with low levels of endogenous NaV current to obviate the need for TTX-resistance mutations. Expression of NaV1.6N or NaV1.6A in ND7/LoNav cells generated NaV currents that differed significantly in voltage-dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared to the corresponding wild-type (WT) channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.

neuroscience↗

bioRxiv2023

Epilepsy-associated SCN2A (Nav1.2) Variants Exhibit Diverse and Complex Functional Properties

Pathogenic variants in neuronal voltage-gated sodium (NaV) channel genes including SCN2A, which encodes NaV1.2, are frequently discovered in neurodevelopmental disorders with and without epilepsy. SCN2A is also a high confidence risk gene for autism spectrum disorder (ASD) and nonsyndromic intellectual disability (ID). Previous work to determine the functional consequences of SCN2A variants yielded a paradigm in which predominantly gain-of-function (GoF) variants cause epilepsy whereas loss-of-function (LoF) variants are associated with ASD and ID. However, this framework is based on a limited number of functional studies conducted under heterogenous experimental conditions whereas most disease-associated SCN2A variants have not been functionally annotated. We determined the functional properties of more than 30 SCN2A variants using automated patch clamp recording to assess the analytical validity of this approach and to examine whether a binary classification of variant dysfunction is evident in a larger cohort studied under uniform conditions. We studied 28 disease-associated variants and 4 common population variants using two distinct alternatively spliced forms of NaV1.2 that were heterologously expressed in HEK293T cells. Multiple biophysical parameters were assessed on 5,858 individual cells. We found that automated patch clamp recording provided a valid high throughput method to ascertain detailed functional properties of NaV1.2 variants with concordant findings for a subset of variants that were previously studied using manual patch clamp. Additionally, many epilepsy-associated variants in our study exhibited complex patterns of gain- and loss-of-function properties that are difficult to classify overall by a simple binary scheme. The higher throughput achievable with automated patch clamp enables study of a larger number of variants, greater standardization of recording conditions, freedom from operator bias, and enhanced experimental rigor valuable for accurate assessment of NaV channel variant dysfunction. Together, this approach will enhance our ability to discern relationships between variant channel dysfunction and neurodevelopmental disorders.

neuroscience↗

bioRxiv2023

Gene Expression Changes in Cultured Reactive Rat Astrocyte Models and Comparison to Device-Associated Effects in the Brain

Implanted microelectrode arrays hold immense therapeutic potential for many neurodegenerative diseases. However, a foreign body response limits long-term device performance. Recent literature supports the role of astrocytes in the response to damage to the central nervous system (CNS) and suggests that reactive astrocytes exist on a spectrum of phenotypes, from beneficial to neurotoxic. The goal of our study was to gain insight into the subtypes of reactive astrocytes responding to electrodes implanted in the brain. In this study, we tested the transcriptomic profile of two reactive astrocyte culture models (cytokine cocktail or lipopolysaccharide, LPS) utilizing RNA sequencing, which we then compared to differential gene expression surrounding devices inserted into rat motor cortex via spatial transcriptomics. We interpreted changes in the genetic expression of the culture models to that of 24 hour, 1 week and 6 week rat tissue samples at multiple distances radiating from the injury site. We found overlapping expression of up to [~]250 genes between in vitro models and in vivo effects, depending on duration of implantation. Cytokine-induced cells shared more genes in common with chronically implanted tissue ([≥]1 week) in comparison to LPS-exposed cells. We revealed localized expression of a subset of these intersecting genes (e.g., Serping1, Chi3l1, and Cyp7b1) in regions of device-encapsulating, glial fibrillary acidic protein (GFAP)-expressing astrocytes identified with immunohistochemistry. We applied a factorization approach to assess the strength of the relationship between reactivity markers and the spatial distribution of GFAP-expressing astrocytes in vivo. We also provide lists of hundreds of differentially expressed genes between reactive culture models and untreated controls, and we observed 311 shared genes between the cytokine induced model and the LPS-reaction induced control model. Our results show that comparisons of reactive astrocyte culture models with spatial transcriptomics data can reveal new biomarkers of the foreign body response to implantable neurotechnology. These comparisons also provide a strategy to assess the development of in vitro models of the tissue response to implanted electrodes.

neuroscience↗

bioRxiv2022

Structural and functional changes of pyramidal neurons at the site of an implanted microelectrode array in rat primary motor cortex

Devices capable of recording or stimulating neuronal signals have created new opportunities to understand normal physiology and treat sources of pathology in the brain. However, it is possible that the initial surgical insertion and subsequent tissue response to implanted electrodes may influence the nature of the signals detected or stimulated. In this study, we characterized structural and functional changes in pyramidal neurons surrounding silicon or polyimide-based electrodes implanted in the motor cortex of rats. Devices were captured in 300 m-thick tissue slices collected at the 1 or 6 week time point post-implantation, and individual neurons were assessed using a combination of whole-cell electrophysiology and 2-photon imaging. We observed disruption of the dendritic arbor of neurons near (<100 m) the device surface at both time points, as well as a significant reduction in spine densities. These effects were accompanied by a decrease in the frequency of spontaneous excitatory post-synaptic currents (sEPSCs), a loss in sag amplitude, and an increase in spike frequency adaptation at the 6 week time point. Interestingly, we also noted a significant increase in filopodial density in neurons surrounding devices. Results were similar for polyimide and silicon-based electrodes. We hypothesize that the effects observed in this study may contribute to the signal loss and instability that often accompany chronically implanted electrodes.

bioengineering↗

bioRxiv2021

Cellular and behavioral effects of altered NaV1.2 sodium channel ion permeability in Scn2aK1422E mice

Genetic variants in SCN2A, encoding the NaV1.2 voltage-gated sodium channel, are associated with a range of neurodevelopmental disorders with overlapping phenotypes. Some variants fit into a framework wherein gain-of-function missense variants that increase neuronal excitability lead to infantile epileptic encephalopathy, while loss-of-function variants that reduce neuronal excitability lead to developmental delay and/or autism spectrum disorder with or without co- morbid seizures. One unique case less easily classified using this binary paradigm is the de novo missense variant SCN2A p.K1422E, associated with infant-onset developmental delay, infantile spasms, and features of autism spectrum disorder. Prior structure-function studies demonstrated that K1422E substitution alters ion selectivity of NaV1.2, conferring Ca2+ permeability, lowering overall conductance, and conferring resistance to tetrodotoxin (TTX). Based on heterologous expression of K1422E, we developed a compartmental neuron model that predicted mixed effects on channel function and neuronal activity. We also generated Scn2aK1422E mice and characterized effects on neurons and neurological/neurobehavioral phenotypes. Dissociated neurons from heterozygous Scn2aK1422E/+ mice exhibited a novel TTX-resistant current with a reversal potential consistent with mixed ion permeation. Cortical slice recordings from Scn2aK1442E/+ tissue demonstrated impaired action potential initiation and larger Ca2+ transients at the axon initial segment during the rising phase of the action potential, suggesting mixed effects on channel function. Scn2aK1422E/+ mice exhibited rare spontaneous seizures, interictal EEG abnormalities, altered response to induced seizures, reduced anxiety-like behavior and alterations in olfactory-guided social behavior. Overall, Scn2aK1422E/+ mice present with phenotypes similar yet distinct from Scn2a knockout models, consistent with mixed effects of K1422E on NaV1.2 channel function. Significance StatementThe early-onset epilepsy variant SCN2A-p.K1422E displays unique biophysical properties in vitro. To model the impact of this rare variant, we generated Scn2aK1422E mice. Neurons from heterozygous Scn2aK1422E/+ mice showed functional deficits similar to the loss-of-function effects observed in the Scn2a haploinsufficiency model, as well as gain-of-function effects specific to the K1422E variant. There is also some overlap in neurobehavioral phenotypes between Scn2aK1422E/+ and Scn2a haploinsufficient mice. However, Scn2aK1422E/+ mice exhibited unique epilepsy-related phenotypes, including epileptiform events and seizures. Scn2aK1422E/+ mice serve as a useful platform to investigate phenotypic complexity of SCN2A-associated disorders.

neuroscience↗

bioRxiv2020

Cryptic prokaryotic promoters explain instability of recombinant neuronal sodium channels in bacteria

Mutations in genes encoding the human brain-expressed voltage-gated sodium (NaV) channels NaV1.1, NaV1.2, and NaV1.6 are associated with a variety of human diseases including epilepsy, autism spectrum disorder, familial migraine, and other neurodevelopmental disorders. A major obstacle hindering investigations of the functional consequences of brain NaV channel mutations is an unexplained instability of the corresponding recombinant complementary DNA (cDNA) when propagated in commonly used bacterial strains manifested by high spontaneous rates of mutation. Here we investigated the cause for instability of human NaV1.1 cDNA. We identified cryptic prokaryotic promoter-like elements that are presumed to drive transcription of translationally toxic mRNAs in bacteria as the cause of the instability, and demonstrated that mutations in these elements can mitigate the instability. Extending these observations, we generated full-length human NaV1.1, NaV1.2, and NaV1.6 plasmids using one or two introns that interrupt the cryptic reading frames along with a minimum number of silent nucleotide changes that achieved stable propagation in bacteria. Expression of the stabilized sequences in cultured mammalian cells resulted in functional NaV channels with properties that matched their parental constructs. Our findings explain a widely observed instability of recombinant neuronal human NaV channels, and we describe re-engineered plasmids that attenuate this problem.

neuroscience↗

bioRxiv2020

Functional and Pharmacological Evaluation of a Novel SCN2A Variant Linked to Early-onset Epilepsy

ObjectiveWe identified a novel de novo SCN2A variant (M1879T) associated with infantile-onset epilepsy that responded dramatically to sodium channel blocker antiepileptic drugs. We analyzed the functional and pharmacological consequences of this variant to establish pathogenicity, and to correlate genotype with phenotype and clinical drug response. MethodsThe clinical and genetic features of an infant boy with epilepsy are presented. We investigated the effect of the variant using heterologously expressed recombinant human NaV1.2 channels. We performed whole-cell patch clamp recording to determine the functional consequences and response to carbamazepine. ResultsThe M1879T variant caused disturbances in channel inactivation including substantially depolarized voltage-dependence of inactivation, slower time course of inactivation, and enhanced resurgent current that collectively represent a gain-of-function. Carbamazepine partially normalized the voltage-dependence of inactivation and produced use-dependent block of the variant channel at high pulsing frequencies. Carbamazepine also suppresses resurgent current conducted by M1879T channels, but this effect was explained primarily by reducing the peak transient current. Molecular modeling suggests that the M1879T variant disrupts contacts with nearby residues in the C-terminal domain of the channel. InterpretationOur study demonstrates the value of conducting functional analyses of SCN2A variants of unknown significance to establish pathogenicity and genotype-phenotype correlations. We also show concordance of in vitro pharmacology using heterologous cells with the drug response observed clinically in a case of SCN2A-associated epilepsy.

neuroscience↗