Biology subjects

George, A. L.

Publications and source records attributed to George, A. L..

At least 19 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

Excitatory Dysfunction and Phenotypic Rescue in a Human Neuronal Model of SCN2A-Related Disorders

SCN2A-related disorders result from pathogenic variants in the gene encoding for the voltage-gated sodium channel Nav1.2. Collectively, these disorders result in variable age of onset epilepsy, autism spectrum disorder, and epileptic encephalopathies. While the mechanisms of haploinsufficiency resulting in autism spectrum disorder have been explored in detail, few studies report the impact of pathogenic missense variants in human neurons. In this work, we combined conventional electrophysiology and high-throughput all-optical electrophysiology assays to analyze the SCN2A p.M1879T pathogenic variant associated with early-onset epilepsy and developmental delay. In both platforms, iPSC-derived excitatory neurons expressing the disease variant showed greater firing at higher stimuli compared to the isogenic control neurons (corrected by CRISPR/Cas9), as well as changes to action potential shape (steeper slope and larger amplitude) with evoked firing. We used machine learning techniques on the optical physiology dataset to classify the two genotypes, finding that sodium channel blocking anti-seizure drugs could restore an isogenic phenotype. This work demonstrates proof of sodium channel blocker efficacy in a human neuronal model of SCN2A-related epilepsy and highlights the power of leveraging high-throughput all-optical electrophysiology for testing drug efficacy.

neuroscience↗

bioRxiv2026

An upstream open reading frame represses translation of the neuronal potassium channel KCNQ2

Upstream open reading frames (uORFs) within the 5-untranslated region (5-UTR) of mRNA transcripts can regulate protein translation. Despite widespread prevalence within the human genome, they remain unidentified for many clinically relevant genes. A gene frequently associated with neonatal-onset epilepsy is KCNQ2, which encodes a neuronal voltage-gated potassium channel subunit that functions to dampen neuronal excitability. Heterozygous loss-of-function pathogenic KCNQ2 variants are known to cause a range of neurodevelopmental disorders and epileptic encephalopathies, but there remains an unmet clinical need for patients harboring these variants. We identified a single uORF in KCNQ2 that is highly repressive of protein translation and demonstrated that mutations disabling the uORF start codon enhance translation of encoded potassium channels. Additionally, we show that adenine base editing of the uORF start codon can weaken ribosome engagement at the uORF and enhance translation of the protein in a neuron-like cell line. This study establishes a previously underexplored regulatory feature for KCNQ2 and highlights the importance of understanding uORFs for clinically relevant genes, both for assessing disease risk and therapeutic potential.

molecular biology↗

bioRxiv2026

Combining automated patch clamp with optogenetics enables selective recording of DRG neurons subtypes

Investigating the neurophysiology of nociception is aided by electrophysiological recording from dorsal root ganglion (DRG) neurons. Because DRG neurons are heterogeneous with overlapping electrophysiological properties, methods to distinguish neuron subtypes are valuable for properly interpreting the measurements and drawing conclusions. Automated patch clamp recording offers an approach for conducting these experiments at higher throughput than conventional recording methods, but identification of neuron subtypes is challenging. We developed a method for recording from acutely isolated mouse DRG neurons using automated patch clamp recording coupled to optogenetic stimulation that was capable of discerning NaV1.8 and TRPV1 expressing neuron subpopulations. This approach can facilitate physiological and pharmacological studies of DRG neurons with potential value in developing and testing targeted analgesic agents.

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

Classification models distinguish functional and trafficking effects of KCNQ1 variants to enhance variant interpretation

Missense mutations compromise protein fitness by altering stability and function, which can lead to various clinical disease states. The potassium ion channel KCNQ1 underlies the majority of congenital long QT syndrome (LQTS) cases, one of the most common genetic arrhythmia syndromes. During genetic testing for LQTS, variants of uncertain significance (VUS) confound diagnosis and clinical management. KCNQ1 protein fitness metrics enable mechanistic classification of variants, directly informing the molecular basis for dysfunction and providing clinical interpretation of variants linked to LQTS and other channelopathies. We developed structure-aware random forest classifier models to predict seven metrics of KCNQ1 fitness, four functional electrophysiology measurements (peak current density, voltage-dependence, gating kinetics), and three trafficking values measured by flow cytometry. Our trained models outperformed AlphaMissense in predicting protein fitness, enhancing interpretation of ClinVar VUS and variants classified as ambiguous by AlphaMissense. We demonstrate the classifiers distinguish benign and pathogenic variants from ClinVar and gnomAD and identify systematic patterns of dysfunction and mistrafficking along the functionally critical S4 helix. Our method advances variant effect prediction with a mechanistic classifier that reliably links missense mutations in KCNQ1 to their specific disease-causing mechanisms. As a resource for precision medicine approaches for LQTS or other KCNQ1 channelopathies, we provide the predictions and scores for all KCNQ1 missense variants across the structured region of the protein.

biophysics↗

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↗

bioRxiv2025

High Throughput Screening Identifies a Small Molecule Trafficking Corrector for Long-QT Syndrome Associated KCNQ1 Variants

Congenital long QT syndrome (LQTS) promotes risk for life-threatening cardiac arrhythmia and sudden death in children and young adults. Pathogenic variants in the voltage-gated potassium channel KCNQ1 are the most frequently discovered genetic cause. Most LQTS-associated KCNQ1 variants cause loss-of-function secondary to impaired trafficking of the channel to the plasma membrane. There are currently no therapeutic approaches that address this underlying molecular defect. Using a high throughput screening paradigm, we identified VU0494372, a small molecule that increases total and cell surface expression, and trafficking efficiency of wildtype (WT) KCNQ1 as well as three LQTS-associated variants. Additionally, 16-hour treatment of cells with VU0494372 increased current density for WT KCNQ1 and the LQTS-associated variant V207M. VU0494372 had no impact on KCNQ1 transcription, degradation, or thermal stability. We identified a potential direct interaction site with KCNQ1 at or near the binding site of the KCNQ1 potentiator ML277. Together, these findings demonstrate that small molecules can increase the expression levels and cell surface trafficking of KCNQ1 and introduce a potential new pharmacological approach for treating LQTS.

biochemistry↗

bioRxiv2025

Machine Learning Resolves Functional Phenotypes and Therapeutic Responses in KCNQ2 Developmental Epileptic Encephalopathy iPSC Models

Pathogenic KCNQ2 variants are associated with developmental and epileptic encephalopathy (KCNQ2-DEE), a devastating disorder characterized by neonatal-onset seizures and impaired neurodevelopment with no effective treatments. KCNQ2 encodes the voltage-gated potassium channel KV7.2, which regulates action potential threshold and repolarization. However, the relationship between KV7.2 dysfunction and abnormal neuronal activity remains unclear. Here, we use human induced pluripotent stem (iPSC)-derived neurons from 5 KCNQ2-DEE patients with pathogenic variants and CRISPR/Cas9-corrected isogenic controls to investigate pathophysiological mechanisms. We identify a common dyshomeostatic enhancement of Ca2+-activated small conductance potassium (SK) channels, which drives larger post-burst afterhyperpolarizations in KCNQ2-DEE neurons. Using microelectrode arrays (MEAs), we recorded over 18 million extracellular spikes from >8,000 neurons during 5 weeks in culture and then applied supervised and unsupervised machine learning algorithms to dissect time-dependent functional neuronal phenotypes that defined both patient-specific and shared firing features among KCNQ2-DEE patients. Our analysis identified irregular spike timing and enhanced bursting as functional biomarkers of KCNQ2-DEE and demonstrated the significant influence of genetic background on phenotypic diversity. Importantly, using unbiased machine learning models, we showed that chronic treatment with the KV7 activator retigabine rescues the disease-associated functional phenotypes with variable efficacy. Our findings highlight SK channel upregulation as a critical pathophysiological mechanism underlying KCNQ2-DEE and provide a robust MEA-based machine learning platform useful for deciphering phenotypic diversity amongst patients, discovering functional disease biomarkers, and evaluating precision medicine interventions in personalized iPSC neuronal models.

neuroscience↗

bioRxiv2025

Characterization of the functional and clinical impacts of CACNA1A missense variants found in neurodevelopmental disorders

CACNA1A encodes the P/Q-type CaV2.1 calcium channels whose function underlies neuronal excitability, presynaptic neurotransmitter release, and Ca2+ signaling in neurons. Pathogenic variants in CACNA1A have been found in individuals with various neurological conditions, including hemiplegic migraine, epilepsy, developmental delay, and ataxia. Clinical presentations can vary significantly between patients, with limited information known about the underlying neurobiology of these different clinical patterns. Adding further complication, prior work on pathogenic missense variants has demonstrated variable impacts on CaV2.1 channel function, sometimes in opposite directions. As such, the relationships between specific coding variants, electrophysiological properties, and clinical phenotypes remain elusive. In this study, we determined the biophysical properties of an allelic series of 42 de novo missense CACNA1A variants discovered in a neurodevelopmental disorder cohort of more than 31,000 individuals, together with the most common eight coding variants found in the general population. We found that all but one de novo variant altered at least one aspect of the channel properties examined, and the majority (70%) of the variants reduced the channel current density. In addition, for variants that encode human CaV2.1 channels (hCaV2.1) with detectable currents, nearly 50% altered how channels respond to membrane potential, while common variations did not significantly change any channel biophysical properties. Coupled with our functional analyses and AlphaMissense prediction, we showed that CaV2.1 missense variants significantly underlie the risk of developmental epileptic encephalopathy. Subsequently, we examined the physiological impact of variant hCaV2.1 using NEURON simulations as an omnibus output of neuronal function and found that abnormal biophysical channel properties have a profound impact on Purkinje cell excitability. Most interestingly, we correlated the clinical phenotype with molecular consequences of missense variants provided by our comprehensive functional analyses and found that distinct CaV2.1 channel molecular function is significantly associated with different clinical outcomes. By analyzing an entire allelic series of CACNA1A de novo changes in a large cohort of individuals with neurodevelopmental disorders, we provide a powerful approach to dissecting the role of missense variants in CACNA1A channelopathy, which in turn may help pave the way for future precision medicine initiatives.

neuroscience↗

bioRxiv2025

Functional profiling of KCNE1 variants informs population carrier frequency of Jervell and Lange-Nielsen syndrome type 2

Congenital long-QT syndrome (LQTS) is most often associated with pathogenic variants in KCNQ1 encoding the pore-forming voltage-gated potassium channel subunit of the slow delayed rectifier current (IKs). Generation of IKs requires assembly of KCNQ1 with an auxiliary subunit encoded by KCNE1, which is also associated with LQTS but causality of autosomal dominant disease is disputed. By contrast, KCNE1 is an accepted cause of recessive type 2 Jervell and Lange-Nielson syndrome (JLN2). The functional consequences of most KCNE1 variants have not been determined and the population prevalence of JLN2 is unknown. Methods: We determined the functional properties of 95 KCNE1 variants co-expressed with KCNQ1 in heterologous cells using high-throughput voltage-clamp recording. Experiments were conducted with each KCNE1 variant expressed in the homozygous state and then a subset was studied in the heterozygous state. The carrier frequency of JLN2 was estimated by considering the population prevalence of dysfunctional variants. Results: There is substantial overlap between disease-associated and population KCNE1 variants. When examined in the homozygous state, 68 KCNE1 variants exhibited significant differences in at least one functional property compared to WT KCNE1, whereas 27 variants did not significantly affect function. Most dysfunctional variants exhibited loss-of-function properties. We observed no evidence of dominant-negative effects. Most variants were scored as variants of uncertain significance (VUS) and inclusion of functional data resulted in revised classifications for only 14 variants. The population carrier frequency of JLN2 was calculated as 1 in 1034. Peak current density and activation voltage-dependence but no other biophysical properties were correlated with findings from a mutational scan of KCNE1. Conclusions: Among 95 disease-associated or population KCNE1 variants, many exhibit abnormal functional properties but there was no evidence of dominant-negative behaviors. Using functional data, we inferred a population carrier frequency for recessive JLN2. This work helps clarify the pathogenicity of KCNE1 variants.

genetics↗

bioRxiv2024

Differential roles of NaV1.2 and NaV1.6 in neocortical pyramidal cell excitability

Mature neocortical pyramidal cells functionally express two sodium channel (NaV) isoforms: NaV1.2 and NaV1.6. These isoforms are differentially localized to pyramidal cell compartments, and as such are thought to contribute to different aspects of neuronal excitability. But determining their precise roles in pyramidal cell excitability has been hampered by a lack of tools that allow for selective, acute block of each isoform individually. Here, we leveraged aryl sulfonamide-based molecule (ASC) inhibitors of NaV channels that exhibit state-dependent block of both NaV1.2 and NaV1.6, along with knock-in mice with changes in NaV1.2 or NaV1.6 structure that prevents ASC binding. This allowed for acute, potent, and reversible block of individual isoforms that permitted dissection of the unique contributions of NaV1.2 and NaV1.6 in pyramidal cell excitability. Remarkably, block of each isoform had contrasting--and in some situations, opposing--effects on neuronal action potential output, with NaV1.6 block decreasing and NaV1.2 block increasing output. Thus, NaV isoforms have unique roles in regulating different aspects of pyramidal cell excitability, and our work may help guide development of therapeutics designed to temper hyperexcitability through selective NaV isoform blockade.

neuroscience↗

bioRxiv2024

Novel Mouse Model of Alternating Hemiplegia of Childhood Exhibits Prominent Motor and Seizure Phenotypes

Pathogenic variants in ATP1A3 encoding the neuronal Na/K-ATPase cause a spectrum of neurodevelopmental disorders including alternating hemiplegia of childhood (AHC). Three recurrent ATP1A3 variants are associated with approximately half of known AHC cases and mouse models of two of these variants (p.D801N, p.E815K) replicated key features of the human disorder, which include paroxysmal hemiplegia, dystonia and seizures. Epilepsy occurs in 40-50% of individuals affected with AHC, but detailed investigations of seizure phenotypes were limited in the previously reported mouse models. Using gene editing, we generated a novel AHC mouse expressing the third most recurrent ATP1A3 variant (p.G947R) to model neurological phenotypes of the disorder. Heterozygous Atp1a3-G947R (Atp1a3G947R) mice on a pure C57BL/6J background were born at a significantly lower frequency than wildtype (WT) littermates, but in vitro fertilization or outcrossing to a different strain (C3HeB/FeJ) generated offspring at near-Mendelian genotype ratios, suggesting a defect in reproductive fitness rather than embryonic lethality. Heterozygous mutant mice were noticeably smaller and exhibited premature lethality, hyperactivity, anxiety-like behaviors, severe motor dysfunction including low grip strength, impaired coordination with abnormal gait and balance, and cooling-induced hemiplegia and dystonia. We also observed a prominent seizure phenotype with lower thresholds to chemically (flurothyl, kainic acid) and electrically induced seizures, post-handling seizures, sudden death following seizures, and abnormal EEG activity. Together, our findings support face validity of a novel AHC mouse model with quantifiable traits including co-morbid epilepsy that will be useful as an in vivo platform for investigating pathophysiology and testing new therapeutic strategies for this rare neurodevelopmental disorder.

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↗

bioRxiv2024

Proteomic Analysis Reveals Trilaciclib-Induced Senescence

Trilaciclib, a CDK4/6 inhibitor, was approved as a myeloprotective agent for protecting bone marrow from chemotherapy-induced damage in extensive-stage small cell lung cancer (ES-SCLC). This is achieved through the induction of a temporary halt in the cell cycle of bone marrow cells. While it has been studied in various cancer types, its potential in haematological cancers remains unexplored. This research aimed to investigate the efficacy of trilaciclib in haematological cancers. Utilizing mass spectrometry-based proteomics, we examined the alterations induced by trilaciclib in the chronic myeloid leukaemia (CML) cell line, K562. Interestingly, trilaciclib promoted senescence in these cells rather than cell death, as observed in acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), and myeloma cells. In K562 cells, trilaciclib hindered cell cycle progression and proliferation by stabilising CDK4/6 and downregulating cell cycle-related proteins, along with the concomitant activation of autophagy pathways. Additionally, trilaciclib-induced senescence was also observed in the non-small cell lung carcinoma cell line (NSCLC), A549. These findings highlight trilaciclibs potential as a therapeutic option for haematological cancers and underscore the need to carefully balance senescence induction and autophagy modulation in CML treatment, as well as in NSCLC. ABSTRACT GRAPHIC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/584620v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@a52d70org.highwire.dtl.DTLVardef@4a1da0org.highwire.dtl.DTLVardef@1df00dborg.highwire.dtl.DTLVardef@1b16758_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Plural molecular and cellular mechanisms of pore domain KCNQ2 encephalopathy

KCNQ2 variants in children with neurodevelopmental impairment are difficult to assess due to their heterogeneity and unclear pathogenic mechanisms. We describe a child with neonatal-onset epilepsy, developmental impairment of intermediate severity, and KCNQ2 G256W heterozygosity. Analyzing prior KCNQ2 channel cryoelectron microscopy models revealed G256 as a node of an arch-shaped non-covalent bond network linking S5, the pore turret, and the ion path. Co-expression with G256W dominantly suppressed conduction by wild-type subunits in heterologous cells. Ezogabine partly reversed this suppression. G256W/+ mice have epilepsy leading to premature deaths. Hippocampal CA1 pyramidal cells from G256W/+ brain slices showed hyperexcitability. G256W/+ pyramidal cell KCNQ2 and KCNQ3 immunolabeling was significantly shifted from axon initial segments to neuronal somata. Despite normal mRNA levels, G256W/+ mouse KCNQ2 protein levels were reduced by about 50%. Our findings indicate that G256W pathogenicity results from multiplicative effects, including reductions in intrinsic conduction, subcellular targeting, and protein stability. These studies provide evidence for an unexpected and novel role for the KCNQ2 pore turret and introduce a valid animal model of KCNQ2 encephalopathy. Our results, spanning structure to behavior, may be broadly applicable because the majority of KCNQ2 encephalopathy patients share variants near the selectivity filter.

neuroscience↗

bioRxiv2023

Longitudinal profiling of the pregnancy plasma proteome through organic solvent precipitation and nano LC-MS/MS

Proteins secreted from maternal, fetal, and placental tissue are vital for processes like placental development, immunotolerance, and fetal growth, and are associated with pregnancy complications, necessitating predictive biomarkers. In this study, we introduce an acetonitrile-based precipitation coupled with solid-phase extraction that addresses limitations of current low throughput blood-based biomarker discovery workflows. Our method is efficient and cost-effective, and identified 433 protein groups, showing specific tissue associations and enrichment in reproductive tissues such as the placenta, breast, and endometrium. Significant alterations in proteins related to hormonal regulation, immune modulation, and placental development throughout gestation were observed. This approach offers comprehensive insights into the circulating pregnancy proteome, but also provides a scalable solution for larger studies for biomarker discovery in the context of pregnancy complications. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/571058v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@eb9840org.highwire.dtl.DTLVardef@158cfc5org.highwire.dtl.DTLVardef@1b0a8d9org.highwire.dtl.DTLVardef@11d448d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗