Biology subjects

Shi, R.

Publications and source records attributed to Shi, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Optimizing Cognitive Phenotyping for Genetic Discovery in Alzheimer's Disease: A Simulation-Based Evaluation of Mini-Mental State Examination-Based Decline Measures

Background: Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder characterized by cognitive decline. The Mini-Mental State Examination (MMSE) is a well-established screening tool that evaluates five cognitive domains with a total maximum score of 30. Although previous studies have suggested a genetic basis for cognitive decline in AD, inconsistent findings have been reported and may stem from variability in phenotypic definitions. Methods: We conducted a simulation study using parameters informed by real-world AD cohorts to model 10-year trajectories of MMSE scores in a sample of 500 individuals. Three types of cognitive decline patterns, including linear, quadratic, and sigmoid, were simulated. We incorporated realistic missing data mechanisms to reflect typical challenges in longitudinal data collection. From the simulated MMSE data, we derived seven distinct phenotypic measures of cognitive decline. Using statistical models that assumed additive genetic effects, we compared the power of these phenotypes across different missing data scenarios to identify the most effective strategies for phenotyping in genetic association studies. Results: Adjusting for baseline MMSE improved the statistical power of measures that do not incorporate it in the definition. Across all simulated scenarios, simpler non-repeated measures, such as "Slope", "dMMSEdt", and "Time-to-MMSE10", consistently outperformed the others. Conclusions: The phenotypic measures "Slope" and "dMMSE/dt" consistently showed strong performance across a range of data distributions, while "Time-to-MMSE10" was particularly effective in cohorts with a higher proportion of individuals experiencing severe cognitive decline. These findings highlight the importance of tailoring the choice of cognitive decline measures to the specific characteristics of the dataset, such as the severity and distribution of decline, to improve the power to detect genetic factors associated with Alzheimer's disease.

bioinformatics↗

bioRxiv2026

Telmisartan alters the transcriptomes of cancer associated fibroblasts and fibrocytes in a solid tumour model

Cancer associated fibroblasts (CAFs) are the main contributors to fibrosis in solid tumours. Fibrosis can lead to increased tumoural stiffness, immune cell exclusion, and poor response to cancer therapies. However, therapeutically depleting CAFs has been shown to increase tumour progression and metastasis in some pre-clinical tumour models. Targeted approaches to inhibit CAF activity and decrease fibrosis without abrogating CAFs altogether may improve the effect of existing cancer therapies. We have previously shown that the blood pressure medication telmisartan decreases tumoural collagen, resulting in improved vascular perfusion, decreased hypoxia, and improved radiation treatment efficacy. To understand how telmisartan influences CAF populations in solid tumours, we conducted single cell RNA sequencing analyses of host cells isolated from tumours in mice treated with telmisartan. Telmisartan modified the transcriptomes of CAF populations, promoting the expression of chemokine signaling while decreasing the expression of extracellular matrix components in myCAFs. Telmisartan decreased expression of collagen transcripts in myCAFs and fibrocytes, indicating that telmisartan does not remove myCAFs but changes the myCAF transcriptome to become less fibrillar. Our work provides further support for using telmisartan to manipulate CAF populations in the solid tumour microenvironment to decrease collagen deposition and render tumours more responsive to current cancer therapies.

cancer biology↗

bioRxiv2026

Nuclear size is genetically controlled and influences cell fate

The proportional scaling between nuclear and cell size was first described more than 150 years ago and is among the most conserved features of cellular organization. Yet the mechanisms that establish this scaling and its physiological significance have remained unresolved. Here, we address both questions by combining image-enabled cell sorting with genome-wide CRISPR screening, transcriptomics and functional analyses. We identify more than 180 regulators of the nuclear-to-cell ratio and show that distinct classes of genes independently control nuclear and cell size. RNA metabolism predominantly regulates nuclear size, whereas protein synthesis and degradation primarily regulate cell size. This supports a model in which differences in macromolecular partitioning between the nucleus and cytoplasm contribute to osmotic regulation of nuclear size together with mechanical constraints imposed by chromatin, the cytoskeleton, and the nuclear envelope. Changes in nuclear size cause widespread transcriptional remodeling that is independent of changes in cell size. Cells with smaller nuclei exhibit reduced PRC2-dependent H3K27 trimethylation, activation of developmental gene-expression programmes and repression of cell-cycle genes. Consistent with these changes, mouse embryonic stem cells with smaller nuclei show an increased propensity to exit pluripotency and initiate differentiation in response to retinoic acid. Together, our findings provide a mechanistic framework for nuclear size scaling and establish nuclear size as a physical regulator of gene expression and cell-state transitions, linking cellular architecture to cell fate.

cell biology↗

bioRxiv2026

Epistasis limits but does not prevent the transfer of mutation-drug resistance mapping across 600 million years of fungal evolution

Whether different pathogens acquire resistance to antimicrobials through the same mutations is a major question in evolution and microbiology. Most antifungal drugs are used to treat infections caused by multiple fungal species, many of which have diverged for millions of years. If the evolution of resistance was to converge onto the same set of mutations across species, knowing the mechanism of resistance in one would allow us to predict and track it in others. The extent of this convergence remains unknown due to the lack of systematic data on resistance mutations. Here, we quantify the conservation of resistance mutations in the cytosine deaminase (CD), a protein responsible for resistance to flucytosine, one of the oldest antifungal drugs. By comparing the crystal structures of this enzyme through 600 My of evolution, we show that the CD structure is highly conserved. We compared the full CD mutational resistance spectrum of resistance from an ascomycete and a basidiomycete. We found that resistance mutations in one ortholog can be used to predict resistance in the other at a high level of accuracy. However, because of epistasis, around 10% of mutations have distinct effects in the two orthologs, which imposes an upper limit to the transferability of the knowledge of resistance mutations from one species to another. Using biochemical assays and by structural characterization of several mutants, we identify distinct mechanisms of epistasis, one important being that the local physiochemical environment of some position has evolved in a way that makes the same substitutions destabilizing or entirely inactivating in an ortholog-specific manner. Our results show that resistance mutations can be conserved in fungi across hundreds of millions of years of evolution but that epistasis eventually limits the accuracy of these predictions.

microbiology↗

bioRxiv2026

Low-dimensional Neural Codes Suppress Neuronal Noise and Extend the Working Memory Duration

Neurons are noisy computational substrates, yet large neural populations achieve reliable computation. What determines the maximal duration that a noisy population can sustain working memory? We study this question with recurrent networks subject to stochastic noise and present three theoretical results. First, networks suppress independent neuronal noise when activity lies on a low-dimensional latent manifold. Second, this structure induces correlated noise across neurons, limiting the downstream information that can be extracted. Third, these effects yield an analytical bound on working memory duration that scales linearly with network size. We test these predictions using large-scale neocortical recordings, and provide a behavioral signature in mice consistent with the theory. Overall, noise suppression constitutes a key functional benefit of low-dimensional neural coding, with which large populations sustain reliable working memory over extended timescales.

neuroscience↗

bioRxiv2026

Predictable adaptive evolution of a phage endolysin through substrate recognition optimization

Bacteriophages release progeny by producing endolysins that degrade the bacterial cell wall. While the frequent horizontal transfer of endolysins suggests substantial evolutionary plasticity, the mechanisms by which these enzymes adapt to new phage-host contexts remain poorly understood. Here, we investigated the evolutionary dynamics and structural mechanisms governing endolysin adaptation following experimental evolution of a chimeric phage generated via heterologous endolysin exchange between phages infecting different hosts. Using replicate experimental evolution and time-resolved PacBio sequencing, we identified a dominant, highly reproducible adaptive trajectory characterized by the stepwise fixation of three key mutations. This constrained mutational order correlated with incremental gains in enzymatic activity, reflecting a rugged yet predictable fitness landscape. High-resolution structural analyses revealed that these substitutions lie exclusively outside the catalytic site; instead, they enhance substrate recognition through electrostatic tuning, optimized hydrophobic packing, and local conformational refinement, resulting in significantly higher binding affinity. While the adaptive trajectory was largely conserved, one replicate followed an alternative path, highlighting the interplay between selection and historical contingency. Adaptation was further shaped by a functional trade-off, whereby increased lytic activity on the novel host was accompanied by reduced activity on the ancestral host, consistent with antagonistic pleiotropy. Genome-wide sequencing additionally identified a compensatory mutation in a lytic transglycosylase, suggesting coordinated evolution of the broader lysis machinery. Together, these results demonstrate that endolysins evolve through reproducible adaptive walks constrained by structure, selection, and trade-offs, providing a mechanistic framework for understanding enzyme evolution and informing rational protein engineering.

evolutionary biology↗

bioRxiv2026

EnzCast: Prediction of Patient-Specific Enzymatic Kinetics through Multi-Modal Deep Learning and Isoform-Resolved Bayesian Inference based on Single-Cell Transcriptomics

Enzyme kinetic parameters underpin mechanistic biology but remain sparse in physiological context. We present EnzCast, a multi-modal framework jointly predicting Km, kcat, kcat/Km, and Ki from protein sequence, 3D structure, substrate chemistry, and experimental conditions, paired with IsoKin, an isoform-resolved Bayesian framework converting EnzCast priors into patient-specific in vivo kinetics. Trained on KinBench, the largest curated kinetics database, task-adaptive EnzCast achieved R2 = 0.413, 0.455, 0.227, and 0.105 for Km, Ki, kcat, and kcat/Km, surpassing all baselines on catalytic tasks. Systematic condition scans recovered compartment-specific pH direction inversion and pathway-level temperature responses. In a 20-patient colorectal cancer single-cell cohort, IsoKin reduced posterior uncertainty by 73.3% and 77.3%, revealing cell-type-specific rewiring. Orthogonal validation--scFEA flux, DepMap essentiality (permutation P = 0.0008) and TCGA survival-- provided mixed but directionally consistent support. Together, EnzCast and IsoKin bridge in vitro prediction, condition-aware biochemical interrogation and patient-resolved in vivo inference.

bioinformatics↗

bioRxiv2026

Neurodegeneration risk variants promote lysosomal TMEM106B fibrilaccumulation

Variants in TMEM106B and GRN, which encode lysosomal proteins, interact through unknown mechanisms to increase the risk of age-related cognitive decline and neurodegeneration. Here, we show that these variants converge on a single molecular intermediate: the cleaved intra-lysosomal fibril core of TMEM106B, a precursor to amyloid fibrils that accumulate in the aging brain. A protein-coding TMEM106B risk variant (p.T185) drives fibril core accumulation by impairing its degradation and GRN risk variants amplify this effect. Mice over-expressing the fibril core develop hallmarks of neurodegeneration, and cryo-electron tomography reveals intra-lysosomal fibrils in cultured neurons, mice, and diseased human brain. In GRN-mutation carriers, in whom fibril burden is greatest, fibrils extrude through ruptured lysosomal membranes. These findings identify intra-lysosomal TMEM106B fibrillization as a convergent neurodegeneration mechanism and potential therapeutic target.

cell biology↗

bioRxiv2026

Proteomic analysis reveals shared biological pathways linking acrolein to biomolecular changes in the acute phase of rat spinal cord injury

Spinal cord injury (SCI) pathology is highly difficult to treat due to substantial heterogeneity in injury presentation and spread, along with unclear mechanisms linking damage to pathology. Damages from injury forces (primary injury) are exacerbated by a series of biochemical events that follow the initial damage and injure additional tissue, known as secondary injury. Reactive aldehydes, such as acrolein, play a key role in propagating secondary injury cascades following SCI. Targeting acrolein after SCI has demonstrated therapeutic potential in limiting injury spread and pathology. However, injury mechanisms linking reactive aldehydes to SCI outcome have not been fully characterized. To gain a more comprehensive understanding of the cellular and molecular mechanisms underlying SCI, we generated proteomic profiles of rat spinal cords 24 h (acute phase) after subjection to SCI, sham injury, saline injection, or acrolein injection. We performed gene set enrichment analysis (GSEA) to characterize proteins and pathways significantly enriched after SCI and acrolein-injection. We then used Translatable Components Regression (TransComp-R), a framework for translating biological signatures across systems, to assess whether acrolein-associated spinal cord signatures can stratify SCI from sham outcomes. Our proteomics analysis revealed 467 differentially expressed proteins (DEPs) between the sham and SCI groups and 7 DEPs between saline and acrolein injection groups. Notably, the complement and coagulation cascades were upregulated in spinal cords subjected to SCI and acrolein injection. Our TransComp-R analysis further demonstrated that acrolein-associated signatures could distinguish SCI from sham conditions. Taken together, our findings suggest that acrolein induces proteomic alterations during the acute phase of SCI and is associated with complement and coagulation cascade activation, among other pathways. Therefore, this study reinforces the notion that understanding the role of acrolein in the acute phase of secondary SCI may be beneficial.

neuroscience↗

bioRxiv2025

Construction of a randomly barcoded insertional mutant library in the filamentous fungus Trichoderma atroviride

Filamentous fungi play key roles in ecosystems, agriculture, biotechnology, symbiosis, and disease, yet the large-scale characterization of gene function in these organisms remains limited by low transformation efficiencies and their multinucleate, syncytial cells, which complicate high-throughput screening strategies. To address the challenge of high-throughput screening in filamentous fungi, we developed methods to construct a genome-wide barcoded insertional mutant library in Trichoderma atroviride, a filamentous fungus widely used as a biocontrol agent against bacterial and fungal plant pathogens. Our strategy leveraged randomly barcoded transfer DNA insertions from plasmid libraries containing hundreds of millions of unique DNA barcodes and a broad host-range drug resistance marker delivered via Agrobacterium tumefaciens into T. atroviride. By optimizing transformation conditions, we achieved up to 600 independent transformants per infection event, resulting in a library of over 31,000 mapped insertions disrupting 7,104 of the 11,863 predicted genes in the T. atroviride genome. This resource establishes a scalable platform for high-throughput functional genomics in filamentous fungi, enabling both fundamental investigations of fungal biology and engineering approaches toward improved medical applications, biotechnology, and sustainable agriculture.

systems biology↗

bioRxiv2025

Design of Tissue-Selective PROTACs Through Recruiting E3 Ligase Scaffolding Protein MAGEA11

Proteolysis targeting chimeras (PROTACs) are an emerging therapeutic modality that induces protein degradation by recruiting E3 ligases. Most reported PROTACs recruit ubiquitously expressed E3 ligases, such as cereblon and the von Hippel-Lindau tumor suppressor. Of the additional 600+ E3 ligases, recruiting those with tissue-restricted expression is attractive for increasing the specificity of PROTACs. To this end, tissue-specific E3 ligases or E3 ligase-associated proteins that can be recruited for targeted protein degradation need to be identified. This work describes the first reported PROTAC that recruits the tissue-specific E3 ligase scaffolding protein MAGEA11. As an initial demonstration, a library of bromodomain and extra-terminal domain (BET)-targeting PROTACs that recruit MAGEA11 was synthesized. The library was screened in osteosarcoma U2OS cells, identifying lead compound 105B. 105B potently degrades BET proteins in U2OS osteosarcoma cell lines (BRD4 DC50 = 0.130 nM, Dmax = 78%) and KYSE180 esophageal squamous cell carcinoma cell lines (DC50 = 40 nM, Dmax = 70%), but shows no degradation in non-cancerous, MAGEA11-deficient HEK293T cells. Mechanistic studies confirmed 105Bs dependence on the ubiquitin-proteasome system and engagement of both MAGEA11 and BRD4. 105B decreased levels of BET-regulated gene products c-Myc, RUNX2, and KRT14; however, improvements are still necessary to affect selective cytotoxicity. This work reports the first example of a PROTAC recruiting a tissue-specific E3 ligase for cancer-restricted degradation of BET proteins and highlights the need for further development of MAGEA11-recruiting degraders.

biochemistry↗

bioRxiv2025

Mechanistic Insights into Cyclic Penta-Adenylate-Mediated Activation of Type III CRISPR Ribonuclease Csm6

Type III CRISPR systems generate cyclic oligoadenylate (cOA, 3 to 6 AMPs) messengers upon detecting viral RNA, activating downstream effectors to defend against viral infection. Although cOA-activated effectors have been extensively characterized, the cA5-specific effectors remained unexplored despite cA5 being among the most abundant cOA species produced during phage infection. Here, we report that Actinomyces procaprae Csm6 (ApCsm6) selectively employs cA5 as its activator. Unlike other characterized Csm6 proteins, ApCsm6 self-limits its ribonuclease activity by degrading cOAs via its HEPN domain, rather than relaying on the CARF domain. Cryo-EM structures of ApCsm6 and its complexes with cA5 and cA6 reveal a homotetrameric assembly, where each monomer binds a single cOA within a composite pocket formed by two tandem CARF-HEPN domains. Binding of cA5, but not cA6, enhances tetramerization and induces large conformational shifts in CARF, which in turn allosterically activates ssRNA cleavage in HEPN. These findings advance our understanding of ligand discrimination and signaling regulation in type III CRISPR immunity. HighlightsO_LIApCsm6 preferentially recognizes cA5 as its activator. C_LIO_LICryo-EM structures of ApCsm6 and its complexes with cA5 or cA6 reveal the structural basis for cA5-selective recognition and allosteric activation. C_LIO_LIApCsm6 acts as a self-limiting ribonuclease by degrading cOAs via its HEPN domain rather than relaying on the CARF domain. C_LI

biochemistry↗

bioRxiv2025

Structure-Guided Development of a Potent BioA Inhibitor ValidatesBiotin Synthesis Inhibition as a Therapeutic Strategy forTuberculosis

Conditionally essential pathways - such as the biotin biosynthesis - represent promising targets for new antibiotics. However, the chemical interrogation of the biotin pathway with an orally effective lead remains elusive, and the preclinical development of biotin inhibitors for mycobacterial infections in vivo is challenging due to the unusually high concentration of biotin in standard mouse models. Structure-guided optimization was applied to develop the first oral lead targeting aminotransferase BioA, a key enzyme in bacterial biotin biosynthesis, resulting in C48, a picomolar inhibitor displaying sub-micromolar MICs against Mycobacterium tuberculosis (Mtb). Mechanism of action was confirmed by biochemical, structural, and genetic studies. C48 demonstrated favorable pharmacokinetics and excellent oral bioavailability resulting in over 39,000-fold improved exposure. We next developed an easy-to-operate, low-biotin mouse model that recapitulates human biotin physiology. C48 significantly reduced Mtb burden in this low-biotin mouse model, providing the first in vivo proof-of-concept for targeting biotin biosynthesis in Mtb.

microbiology↗

bioRxiv2025

Impact Induces Phagocytic Defect in Reactive Microglia

We have developed traumatic brain injury (TBI)-on-a-chip in vitro models using primary microglia and neuronal networks and recorded the molecular and cellular changes following impact to represent impact injury. Using a pH-responsive amyloid {beta} (A{beta}pH), we showed that microglial phagocytosis was reduced at 7 days post-impact on the chip. Simultaneously, neurons increased their uptake of A{beta}, and decreased neuronal firing frequency at 7 days post-impact based on electrophysiological recordings. Given the importance of lipid metabolism in brain trauma and neurodegeneration, the lipidome secreted by impacted cells was analyzed to understand changes in cellular processes. Interestingly, many lipid species from the sphingomyelin, glycerophospholipid, and phosphatidylserine classes were significantly affected by impact, which are known to play important roles in the resolution of neuroinflammation and the pathogenesis of neurodegeneration.

neuroscience↗

bioRxiv2025

mRNABench: A curated benchmark for mature mRNA property and function prediction

Messenger RNA (mRNA) is central in gene expression, and its half-life, localization, and translation efficiency drive phenotypic diversity in eukaryotic cells. While supervised learning has widely been used to study the mRNA regulatory code, self-supervised foundation models support a wider range of transfer learning tasks. However, the dearth and homogeneity of standardized benchmarks limit efforts to pinpoint the strengths of various models. Here, we present O_SCPLOWMC_SCPLOWRNABO_SCPLOWENCHC_SCPLOW, a comprehensive benchmarking suite for mature mRNA biology that evaluates the representational quality of mature mRNA embeddings from self-supervised nucleotide foundation models. We curate ten datasets and 59 prediction tasks that broadly capture salient properties of mature mRNA, and assess the performance of 18 families of nucleotide foundation models for a total of 135K experiments. Using these experiments, we study parameter scaling, compositional generalization from learned biological features, and correlations between sequence compressibility and performance. We identify synergies between two self-supervised learning objectives, and pre-train a new Mamba-based model that achieves state-of-the-art performance using 700x fewer parameters. O_SCPLOWMC_SCPLOWRNABO_SCPLOWENCHC_SCPLOW can be found at: https://github.com/morrislab/mRNABench.

genomics↗

bioRxiv2025

Unveiling the biochemical catalysis and regulatory mechanisms of the E3-independent E2 enzyme hUBE2O

UBE2O, an E3-independent E2 ubiquitin-conjugation enzyme, directly engages substrates to mediate Ubiquitin conjugation and ligation. Despite its critical role in ubiquitination of multiple substrates, the catalytic and regulatory mechanisms of Homo sapiens UBE2O (hUBE2O) remain incompletely understood. Here, combining domain truncation, systematic mutagenesis and well-designed biochemical approaches, we demonstrate that hUBE2O mediates both mono- and polyubiquitination through a unique catalytic architecture. While none cysteines function for hUBE2Os E3 activity, the coiled-coil (CC) and C-terminal regulatory (CTR) domains maintain catalytic competence, with the N-terminal regions impose activity constraints. Interestingly, hUBE2O activity is refractory to its self-ubiquitination and phosphorylation state. Instead, specific non-cysteine residues (H939, T995, S1042A, S1046A, S1060A and H1130) emerge as critical regulators of substrate selectivity and catalytic optimization. Surprisingly, zinc ions emerge as potent allosteric inhibitors that bind cysteines of hUBE2O, sterically occluding the access of catalytic site C1040 to Ubiquitin. Our findings reveal that hUBE2O-mediated E3-independent ubiquitination is governed by dynamic interdomain cooperation and allosteric modulation, establishing a mechanistic framework for understanding non-canonical ubiquitination and informing the development of targeted hUBE2O modulators.

biochemistry↗

bioRxiv2025

The Pathological Role and Therapeutic potential of ALDH2 in acrolein detoxification Following Spinal Cord Injury in Mice

Oxidative stress and lipid peroxidation-derived aldehydes, such as acrolein, play a central role in the pathology of spinal cord injury (SCI) and have emerged as promising therapeutic targets. Mitochondrial aldehyde dehydrogenase-2 (ALDH2) is a key oxidoreductase responsible for detoxifying reactive aldehydes. Pharmacological activation of ALDH2 using Alda-1, a selective agonist, has been shown to reduce aldehyde accumulation, alleviate inflammation, and enhance functional recovery in experimental SCI models. However, approximately 8% of the global population carries the ALDH2*2 mutation, which severely impairs this detoxification pathway. In this study, we used a transgenic ALDH2*2 mouse model to investigate the role of ALDH2 in SCI pathology. This model mimics the human ALDH2*2 condition, allowing us to examine the impact of impaired aldehyde clearance on acrolein accumulation and its pathological consequences. We modulated endogenous aldehyde detoxification through both genetic deficiency and pharmacological activation with Alda-1. Our results showed that ALDH2 deficiency led to significantly elevated acrolein levels, which were associated with increased microglial activation, cytokine storm, neuronal loss, demyelination, and tissue damage compared to wild-type (WT) mice. Treatment with Alda-1 enhanced ALDH2 activity and significantly reduced acrolein levels in both ALDH2*2 and WT mice from 2 to 28 days post-SCI. This was accompanied by reduced inflammation, improved preservation of myelin, and marked improvements in locomotor and sensory function, especially in ALDH2*2 mice. Notably, even beyond the traditionally ideal treatment window, Alda-1 treatment remained effective in promoting recovery, particularly in motor function and to a greater extent in ALDH2*2 mice. Our study comprehensively evaluated ALDH2s role in SCI by both genetically impairing and pharmacologically enhancing its activity, highlighting ALDH2 as a critical modulator of acrolein-mediated damage and suggesting its potential as a therapeutic target, especially for individuals with the ALDH2*2 mutation.

neuroscience↗

bioRxiv2025

Gene therapies alleviate absence epilepsy associated with Scn2a deficiency in DBA/2J mice

Mutations in the voltage-gated sodium channel gene SCN2A, which encodes the NaV1.2 channel, cause severe epileptic seizures. Patients with SCN2A loss-of-function (LoF) mutations, such as protein-truncating mutations, often experience later-onset and drug-resistant epilepsy, highlighting an urgent unmet clinical need for new therapies. We previously developed a gene-trap Scn2a (Scn2agt/gt) mouse model with a global NaV1.2 reduction in the widely used C57BL/6N (B6) strain. Although these mice display multiple behavioral abnormalities, EEG recordings indicated only mild epileptiform discharges, possibly attributable to the seizure-resistant characteristics associated with the B6 strain. To enhance the epileptic phenotype, we derived congenic Scn2agt/gt mice in the seizure-susceptible DBA/2J (D2J) strain. Notably, we found that these mice exhibit prominent spontaneous absence seizures, marked by both short and long spike-wave discharges (SWDs). Restoring NaV1.2 expression in adult mice substantially reduced their SWDs, suggesting the possibility of SCN2A gene replacement therapy during adulthood. RNA sequencing revealed significant alterations in gene expression in the Scn2agt/gt mice, in particular a broad downregulation of voltage-gated potassium channel (KV) genes, including KV1.1. The reduction of KV1.1 expression was further validated in human cerebral organoids with SCN2A deficiency, highlighting KV1.1 as a promising therapeutic target for refractory seizures associated with SCN2A dysfunction. Importantly, delivery of exogenous human KV1.1 expression via adeno-associated virus (AAV) in D2J Scn2agt/gt mice substantially reduced absence seizures. Together, these findings underscore the influence of mouse strain on seizure severity and highlight the potential of targeted gene therapies for treating SCN2A deficiency-related epilepsies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/657652v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@35374eorg.highwire.dtl.DTLVardef@1c22197org.highwire.dtl.DTLVardef@2b4582org.highwire.dtl.DTLVardef@e06e68_HPS_FORMAT_FIGEXP M_FIG C_FIG In briefScn2a deficiency leads to absence seizures in D2J mice and neuronal hyperexcitability with compensatory KV reduction; restoring NaV1.2 or introducing human KV1.1 reduces seizure burden. HighlightsO_LIScn2a deficiency induces robust absence seizures in the DBA/2J but not the C57BL/6N strain. C_LIO_LICortical neurons in adult DBA/2J mice with Scn2a deficiency exhibit intrinsic hyperexcitability. C_LIO_LISevere Scn2a deficiency leads to downregulation of multiple potassium channel genes. C_LIO_LIGenetic restoration of NaV1.2 expression alleviates spike-wave discharges (SWDs). C_LIO_LIAAV-mediated human KV1.1 delivery substantially reduced absence seizures, demonstrating the therapeutic potential of targeted gene therapy. C_LI

neuroscience↗