Cell biology
Abstract
Pathogenic SCN2A loss-of-function (LoF) variants can cause severe, refractory epilepsy with limited treatment options. Existing Scn2a-LoF mouse models exhibit few spontaneous seizures, limiting the dynamic range needed to assess therapeutic efficacy. We therefore generated gene-trap Scn2a-deficient mice on the seizure-prone DBA/2J background. These mice experienced hundreds of spontaneous absence seizures per day, characterized by concurrent behavioral arrest and EEG spike–wave discharges (SWDs). Partial restoration of Scn2a expression reduced seizure burden. Electrophysiological analysis revealed impaired action potential waveforms yet increased repetitive firing, alongside coordinated downregulation of voltage-gated potassium channel genes, potentially contributing to hyperexcitability. Given the broad potassium channel downregulation and prior efficacy of KCNA1 gene therapy in refractory seizure models, we selected KV1.1 as a potential therapeutic target and used a drug-controllable adeno-associated virus (AAV) to express human KCNA1. We found that this intervention normalized elevated neuronal firing and reduced spontaneous seizure burden in Scn2a-deficient mice. Extending these findings to a human cell-based platform, AAV-mediated KCNA1 expression also reduced neuronal firing in human cortical organoids carrying a SCN2A-truncating variant identified in patients. Together, these findings provide proof of principle for drug-controllable KCNA1 gene therapy as a promising treatment avenue for epilepsy related to SCN2A LoF variants.
Authors
Zaiyang Zhang, Jingliang Zhang, Xiaoling Chen, Brody A. Deming, Shivam Kant, Purba Mandal, Harish Kothandaraman, Phillip J. SanMiguel, Manasi S. Halurkar, Akila D. Abeyaratna, Morgan J. Robinson, Iuliia Vitko, Ronald P. Gaykema, Chongli Yuan, Nadia A. Lanman, Matthew T. Tegtmeyer, Dan Wang, Guangping Gao, Riyi Shi, Edward Perez-Reyes, Yang Yang
Abstract
Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor critical for host defense against microbial infections. To prevent aberrant activation, nuclear cGAS is restrained by nucleosome tethering or by barrier-to-autointegration factor–mediated restriction of its access to DNA. However, its physiological roles beyond enzymatic suppression in the nucleus remain poorly understood. Here, we present a genome-wide map of cGAS chromatin localization in human cells and unexpectedly find its binding beyond nucleosome tethering, where cGAS is recruited to chromatin by AP-1 transcription factors via a potentially previously uncharacterized transactivation domain. Integrated CUT&RUN, RNA-seq, and metabolomics analyses reveal that cGAS transcriptionally regulates cholesterol homeostasis. Mechanistically, cGAS promotes LGALS3 transcription, which inhibits ACAA2 activity to suppress fatty acid oxidation and acetyl-CoA production, thereby lowering intracellular cholesterol levels. Reduced cholesterol lowers plasma membrane phosphatidylinositol [3,4,5] triphosphate [PI(3,4,5)P3] levels and decreases mTOR complex 2 (mTORC2) activation. In contrast, cGAS depletion results in cholesterol accumulation and elevated PI(3,4,5)P3, driving mTORC2/Akt signaling, promoting xenografted tumor growth, and conferring resistance to DNA-damaging agents. In vivo, the elevated hepatic cholesterol levels observed in cGas–/– mice were largely reversed by inhibition of Akt but not STING, whereas cGAS deficiency had minimal effects on lung physiology. Collectively, our findings uncover an unexpected role for nuclear cGAS in orchestrating a transcriptional program that links lipid metabolism to cell proliferation, stress response, and cholesterol homeostasis in liver.
Authors
Yu Deng, Matthew J. Niederhuber, Zhichuan Zhu, Austin J. Hepperla, Le Yu, Quentin Hahn, Joshua Hartsell, Tooba Rashid, Piotr A. Mieczkowski, Hemant Kelkar, Jeremy M. Simon, Yanqiong Zhang, Alessandra Livraghi-Butrico, Meredith J. Zeller, Joseph G.C. Kennedy, Jazmin F. DePriest, Jiong Li, Yan Zhang, Xing Qiu, H. Ümit Kaniskan, Jian Jin, Corbin D. Jones, Laura E. Herring, Douglas H. Phanstiel, Xiaojing Liu, Jesse R. Raab, Brian D. Strahl, Pengda Liu
Abstract
Transmembrane protein 175 (TMEM175), implicated in Parkinson’s disease (PD) risk, has been variably proposed to function as a background K+ “leak” channel or a proton-activated proton channel in lysosomes. Here, we identified the rare TMEM175 variant p.L275R in a patient with early onset PD. Electrophysiological analyses revealed that TMEM175-L275R mutant proteins retain normal lysosomal expression and K+ “leak” activity, but specifically lose proton- and agonist-dependent activation gating. In skin fibroblasts isolated from the patient with PD harboring the monoallelic p.L275R mutation (TMEM175+/L275R), and in derived induced pluripotent stem cells and induced dopaminergic neurons, lysosomal H+ flux, acidification, and degradation were impaired. These lysosomal defects were corrected upon genome editing to produce the isogenic WT allele (+/+Cor), but appeared when the mutant allele was introduced onto a WT background (+/L275RMut). Pathological α-synuclein aggregation was facilitated in patient-derived (+/L275R) and mutation-introduced (+/L275RMut) neurons. Consistent with these genetic findings, pharmacological activation of TMEM175 with small-molecule agonists restored lysosomal functions and reduced α-synuclein aggregation in TMEM175+/L275R neurons. In in-vivo studies, equivalent-site–mutation knock-in mice demonstrated synucleinopathy and PD-like motor phenotypes. Collectively, our work provides genetic and pharmacological evidence that severe loss-of-function mutations may contribute causally to the onset age and pathogenesis of PD and identifies lysosomal proton channels and transporters as potential therapeutic targets for PD intervention.
Authors
Xiaotong Yang, Ping Li, Man Lv, Lingyong Wang, Ruyi Shan, Zikai Jiang, Danzhen Chen, Fangqian Huang, Weijie Cai, Liding Zhao, Zuoyou Guo, Xinhui Chen, Zhidong Cen, Yichang Wang, Wei Yang, Xinran Li, Wei Luo, Lin Yang, Meiqin Hu, Haoxing Xu
Abstract
A significant challenge for improving the efficacy of immune checkpoint blockade (ICB) therapy is the concurrent activation of Tregs, which can dampen antitumor responses. Thus, elucidating the mechanisms that maintain Treg-suppressive function within the tumor microenvironment is critical. Here, we identified an accumulation of N-acetyltransferase 10–modified (NAT10-modified) RNA, particularly in NAT10+FOXP3+ Tregs, in human non–small cell lung cancer. Genetic ablation of Nat10 in tumor-infiltrating (TI) Tregs, or pharmacological inhibition of NAT10 expression, significantly reduced the PD-1+ Treg/CD8+ T cell ratio. These effects were accompanied by an increase in IFN-γ+ Tregs, a reduction in the immunosuppressive activity of Tregs, and inhibition of tumor progression. Mechanistically, NAT10 promotes TI Treg infiltration and activation independently of its RNA-modifying function by directly interacting with FOXP3 and chromatin, modulating local 3D chromatin contacts. Combined administration of anti–PD-1 antibodies and a NAT10 inhibitor further impaired TI Treg–mediated immunosuppression, leading to enhanced tumor control. Our findings demonstrate that NAT10 is a key regulator of TI Treg fragility and function and that its inhibition may enhance the therapeutic efficacy of ICB.
Authors
Xuewei Qi, Dan Cui, Xiao Li, Wenfan Fu, Handong Ma, Wenting Guo, Jiabao Wang, Jiaxi Song, Jiaxin Ma, Jing Yang, Haoyu Yu, Yuting Hong, Qian Wang, Susu Luo, Jia Wang, Baixing Wu, Shanshan Li, Hao Cheng, Min Cheng, Bin Li, Kaiming Zhang, Jia Li, Fengyin Li
Abstract
D1-type receptor (D1R)-mediated dopaminergic signaling within the nucleus accumbens shell (NAcSh) is essential for forming adaptive circuit changes that embed persistent memories associated with drug seeking and craving. While D1R is expressed in both NAcSh neurons and astrocytes, the specific contribution of astrocytic D1R to drug-related circuit plasticity remains poorly understood. Here, we demonstrated in mouse NAcSh slices that D1R agonists increased astrocytic Ca2+ activity, an effect that was attenuated by astrocyte-specific D1R knockdown. Furthermore, selective knockdown of astrocytic D1R in the NAcSh prior to cocaine self-administration inhibited cocaine-induced generation of silent synapses, thereby hampering the associated remodeling of NAcSh circuits. Behaviorally, knockdown of NAcSh astrocytic D1R expedited the extinction of cocaine seeking and reduced cue-induced reinstatement. These results identified astrocytic D1R as a fundamental component of NAcSh dopamine signaling during cocaine experience that remodeled synaptic connections and neural networks underlying drug seeking and relapse.
Authors
Shi Yan, Qize Li, Yao Wang, Wanqiu Chen, Zhong Chen, Alexander K. Zinsmaier, Paloma R. Huguet, Zhengsong Lu, Xiguang Qi, Zheng Xu, Yi Han, Gouri Sharma, Charles Wang, Eric J. Nestler, Oliver M. Schlüter, Yan Dong
Abstract
Golgin-45 (G45) is a coiled-coil protein that transits reversibly between the Golgi and nucleus. In the Golgi, G45 serves as a tether for effectors of a pro-tumorigenic secretory program, but its nuclear role remains unclear. Here, we report that, upon entering the nucleus, G45 functions as a basic leucine zipper transcription factor. From genomic mapping, RNA-sequencing, and functional validation studies, we identify G45-binding sites and show that nuclear G45 can function as a transcriptional activator or repressor, a versatility enabled by phase separation into nuclear bodies enriched in transcriptional co-regulators. In lung cancer models, Golgi-docking reinforces, whereas nuclear translocation opposes, pro-tumorigenic activity of G45. Nuclear G45 incorporates into dimeric transcription factors that silence pro-tumorigenic secreted cargos. Epigenetic events in lung cancer degrade nuclear G45 and sequester G45 in the Golgi, which unleashes malignant secretion. Thus, inter-organelle transitions govern a G45-dependent secretory axis that drives lung cancer progression.
Authors
Chao Wu, Abhishek Kumar, Shike Wang, Weina Zhao, Yuanxin Xi, Jing Wang, Abhinav K. Jain, William K. Russell, Hou-fu Guo, Xiaochao Tan, Jonathan M. Kurie
Abstract
Chronic myelomonocytic leukemia (CMML) is a hematological neoplasm characterized by expansion of malignant monocytes. However, as a stem cell neoplasm, the disease clone comprises most blood lineages, including granulocytes. To investigate their pathogenic contribution, we performed extensive phenotypic, transcriptomic and functional characterization of CMML granulocytes. These cells exhibited reduced granularity and impaired maturation, the latter of which is associated with poor prognosis. Functionally, CMML granulocytes displayed reduced phagocytic capacity and impaired NETosis, together with aberrant gene expression programs. Notably, RETN, encoding the chemokine resistin, was uniquely upregulated in CMML granulocytes. Accordingly, resistin levels were 10-fold higher in CMML plasma, correlating with inferior survival. Ex vivo exposure to resistin directly blocked monocyte maturation and macrophage differentiation, partly via TLR4 interaction, recapitulating the classical monocytosis repartitioning phenotype that characterizes CMML. Resistin-treated monocytes upregulated gene signatures related to immune suppression and myeloid-derived suppressor cell phenotype. Consistent with these findings, CMML patients displayed higher Treg proportions and Th2/Th1 ratios. Furthermore, we demonstrated that resistin directly skews the Th2/Th1 ratio via binding to monocytes. Thus, CMML granulocytes produce high levels of resistin, which contributes to defective monocyte maturation and induction of myeloid-derived suppressor cell features in monocytes, thereby fostering an immune-suppressive state in this disease.
Authors
Nathan J. Hull, Rachel Cant, Laura A. Guest, Yu-Hung Wang, Kristian Gurashi, Roberto Paredes, Carlos Rivera Rivera, Revekka Lefkati, Hector Huerga Encabo, Chien-Chin Lin, Hwei-Fang Tien, Robert Sellers, Sudhakar Sahoo, Dominique Bonnet, Eric Solary, Dorothee Selimoglu-Buet, Daniel H. Wiseman, Kiran Batta
Abstract
Filamin C (FLNC) is an essential cytoskeletal protein required for cardiomyocyte structural integrity and mechanotransduction. Truncating FLNC variants cause haploinsufficiency associated with dilated and arrhythmogenic cardiomyopathies, yet no therapies currently address the underlying defect of FLNC deficiency. Gene replacement is challenging because the approximately 8.2-kb FLNC coding sequence exceeds the packaging capacity of conventional adeno-associated virus (AAV) vectors. Here, we engineered a triple-AAV strategy optimized for full-length FLNC reconstitution in the heart. Screening in HEK293 cells identified GP41-1 and IMPDH as a compatible orthogonal pair for efficient full-length FLNC reconstitution. In tamoxifen-inducible, cardiomyocyte-specific Flnc knockout mice, systemic delivery of optimized MyoAAV2A vectors restored full-length FLNC expression and re-established sarcomeric localization following cardiomyocyte FLNC depletion. A total vector dose of 1 × 10¹³ genome copies/kg restored FLNC to approximately endogenous abundance, preserved systolic function and ventricular dimensions, limited cardiac enlargement, attenuated fibrotic remodeling and prevented premature death. FLNC replacement also attenuated pathological transcriptional remodeling and compensatory changes in cytoskeletal and junction-associated proteins. These findings demonstrate that systemic split-intein-mediated reconstitution of full-length FLNC can preserve cardiac structure and function in vivo, providing preclinical support for a mutation-independent therapeutic strategy for FLNC-associated cardiomyopathy.
Authors
Zhen Jiang, Wei Feng, Zeyu Chen, Julius Bogomolovas, MengChen Li, Mao Ye, Yusu Gu, Xiaohai Zhou, Ju Chen
Abstract
Diabetic retinopathy features early glial stress and neurovascular dysfunction, yet Müller glial programs linking inflammatory cues to oxidative injury remain unclear. We identify a spatially clustered interferon-stimulated gene 15 (ISG15)+ Müller glial subtype expanded in streptozotocin-induced diabetic mice. High glucose alone did not increase basal free ISG15 or detectable ISGylation (ISG15-dependent protein modification) in primary Müller glia, whereas conditioned medium from high glucose–exposed microglia induced both, recapitulated by IFN-β and attenuated by type I interferon receptor 1 inhibition. In vivo, Ifnb1 RNAscope signals were associated with microglia, retinal Ifnb1 expression increased in diabetic retinas, and microglial depletion reduced Ifnb1 expression and limited the expansion of ISG15+ Müller glia. Müller glia–specific Isg15 deletion preserved blood–retinal barrier integrity, reduced acellular capillaries and Evans blue leakage, and mitigated oxidative stress, lipid peroxidation, and glutathione redox imbalance. Loss of ISG15 or disruption of ISGylation blunted interferon-driven oxidative stress in Müller glia and reduced Müller glia–conditioned medium–dependent endothelial migration and tube formation. Mechanistically, IFN-β promoted peroxiredoxin 6 (PRDX6) ISGylation and proteasome-dependent degradation without altering Prdx6 transcripts. Thus, microglial IFN-β promotes an ISG15+ Müller glial subtype that compromises antioxidant defense and promotes early barrier injury in diabetic retinopathy.
Authors
Tian Niu, Yan Jiang, Xindan Xing, Qian Zhu, Xinyi Liu, Kangjia Lv, Junwei Fang, Kun Liu
Abstract
Osteoarthritis (OA) is the most common degenerative joint disease. However, the molecular mechanisms underlying OA-induced cartilage degeneration remain elusive. Here, we identify IFN regulatory factor 2 binding protein 2 (IRF2BP2) as an important regulator of chondrocyte differentiation and articular cartilage regeneration. Irf2bp2 deficiency promoted chondrogenesis and inhibited chondrocyte terminal differentiation by transcriptionally upregulating the key chondrogenic marker SRY-box transcription factor 9 (SOX9), inducing its ectopic expression in the hypertrophic zone. Cartilage-specific KO of Irf2bp2 elevated SOX9 levels, attenuated OA progression in the destabilization of the medial meniscus (DMM) model, and enhanced cartilage regeneration and hyalinization in the microfracture model. Mechanistically, IRF2BP2 bound to and stabilized Fos-related antigen 2 (FOSL2), an activator protein 1 (AP-1) family member, thereby facilitating FOSL2/c-Jun complex formation and repressing SOX9 expression. Adeno-associated virus–mediated (AAV-mediated) FOSL2 overexpression reversed the protective phenotype observed in Irf2bp2-deficient mice. These findings establish the Hippo/IRF2BP2/FOSL2/SOX9 regulatory axis as critical for cartilage regeneration. Importantly, AAV-mediated IRF2BP2 knockdown, pharmacological targeting of the IRF2BP2/AP-1 axis using T-5224, and delivery of a peptide that disrupts IRF2BP2-FOSL2 binding via intra-articular injection all preserved cartilage integrity, offering potential therapeutic strategies for OA and cartilage regeneration.
Authors
Hao Zhou, Tiantian Lu, Zhuoqun Gao, Can Cui, Zhiqiang Fang, Yu You, Jing Jiang, Jinjin Xu, Fang Wei, Jing Yu, Hu Zhou, Jinsong Li, Jian Luo, Lei Zhang, Yunyun Jin
No posts were found with this tag.
Copyright © 2026 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)