IFI204 Drives Gasdermin D–Mediated Mitochondrial Permeabilization to Amplify Neuronal Pyroptosis in Ischemic Stroke

IFI204 Drives Gasdermin D–Mediated Mitochondrial Permeabilization to Amplify Neuronal Pyroptosis in Ischemic Stroke | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article IFI204 Drives Gasdermin D–Mediated Mitochondrial Permeabilization to Amplify Neuronal Pyroptosis in Ischemic Stroke Pengfei Xu, Nan Shen, Tian Qiu, Mingyue Zhou, Rui Li, Chunrong Tao, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8641308/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Gasdermin D (GSDMD)–mediated pore formation on mitochondrial membranes is known to exacerbate pyroptosis. The cytosolic DNA sensor interferon activated gene 204 (IFI204) can activate the inflammasome to induce pyroptosis. However, whether and how IFI204 regulates mitochondrial membrane permeabilization to drive pathological outcomes in ischemic stroke remains unclear. Here, using a mouse model of middle cerebral artery occlusion (MCAO), we demonstrate that IFI204 was predominantly expressed in neurons and increased to peak at 24 hours after ischemic injury. Neuron-specific deletion of IFI204 alleviated cerebral infarction, reduced neuronal degeneration, and restored long-term sensorimotor coordination and cognitive function. These protective effects correlated with attenuated neuronal pyroptosis and mitochondrial dysfunction, as evidenced by decreased levels of GSDMD N-terminal fragment (GSDMD-N) and reduced mitochondrial colocalization. Conversely, adeno-associated virus-mediated re-expression of IFI204 in knockout mice restores these pathological features. In vitro, IFI204 is both necessary and sufficient to trigger this cascade. Transcriptomic profiling revealed a significant downregulation of the stimulator of interferon genes (STING) within the NOD-like receptor signaling pathway in IFI204-deficient neurons. Mechanistically, glutathione S-transferase (GST) pull-down assays confirmed a direct interaction between the pyrin domain (PYD) of IFI204 and STING. This interaction triggers caspase-1 activation and GSDMD cleavage, generating GSDMD-N, which subsequently forms pores specifically on mitochondrial membranes. These pyroptotic pores disrupted mitochondrial integrity, exacerbating dysfunction, and facilitating the cytosolic release of mitochondrial DNA (mtDNA), cytochrome c, and aconitase 2. Notably, the released mtDNA further activated IFI204, establishing a pathogenic feed-forward cycle that exacerbates mitochondrial damage and inflammatory neuronal death. Genetic ablation of STING partially abrogated the pyroptosis-promoting effect of IFI204. Collectively, these findings demonstrate that IFI204-driven cytosolic mtDNA sensing underlies a neuronal inflammatory mechanism responsible for pyroptosis and mitochondrial damage in ischemic stroke. IFI204 Pyroptosis GSDMD Mitochondrial DNA (mtDNA) Ischemic stroke Neuroinflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Ischemic stroke remains a leading cause of disability and mortality worldwide. Recanalization therapy constitutes the cornerstone of acute stroke management; however, despite expanded therapeutic time windows and advances in endovascular techniques, its effectiveness is limited by futile recanalization and reperfusion injury, resulting in suboptimal outcomes in a subset of patients [ 1 ] . Neuroinflammation is central to post-stroke pathology and represents a dynamically initiated and persistently sustained response to ischemic insult. This self-amplifying inflammatory cascade not only exacerbates secondary damage but also impairs acute-phase tissue repair, thereby representing a promising therapeutic target [ 2 ] . The innate immune system plays a pivotal role in orchestrating neuroinflammation and programmed cell death by initiating cerebral defense responses through the recognition of damage-associated molecular patterns (DAMPs) [ 3 ] . This immune surveillance is not restricted to specialized immune cells; neurons also express functional pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and NOD-like receptors (NLRs), which detect these danger signals and trigger inflammatory storm [ 4 ] . Pyroptosis, a form of regulated lytic cell death, represents a canonical downstream outcome of PRRs activation. This process is executed through gasdermin D (GSDMD)-mediated perforation of the plasma membrane, leading to cell lysis and release of DAMPs and inflammatory cytokines [ 5 ] . Clinically, elevated plasma levels of pyroptosis-linked cytokines, including interleukin 1 beta (IL-1β) and interleukin 18 (IL-18), correlate with neurological deficit severity and predict poor outcomes in stroke patients [ 6 ] . In animal models, GSDMD, the executor of pyroptosis, localizes to the plasma membrane of vulnerable neurons, indicating sites of cell rupture [ 7 ] . Emerging evidence demonstrates that the GSDMD N-terminal fragment (GSDMD-N) exhibits a stronger binding affinity for mitochondrial cardiolipin than for plasma membrane phospholipids [ 8 ] , and localizes to mitochondria prior to translocation to the plasma membrane [ 9 ] . The preferential mitochondrial localization of GSDMD-N explains why mitochondrial injury precedes plasma membrane rupture and establishing mitochondrial dysfunction as an early hallmark of pyroptosis. Subsequent GSDMD-mediated mitochondrial permeabilization results in the release of multiple DAMPs, including mitochondrial DNA (mtDNA), adenosine triphosphate (ATP), cytochrome c (Cyto c), and reactive oxygen species (ROS). Among these, mtDNA functions as a potent DAMP. Its hypomethylated CpG motifs, which resemble those found in bacterial DNA, enable mtDNA to activate innate immune pathways such as TLRs and inflammasomes [ 10 , 11 ] . As a result, a self-amplifying loop is formed, linking cellular damage to sustained inflammatory signaling. Although the release of mtDNA following a stroke has been well-documented [ 12 , 13 ] , it remains unclear whether neuronal mtDNA release is directly mediated by GSDMD on mitochondrial membranes. Interferon activated gene 204 (IFI204), along with its human homolog interferon gamma-inducible protein 16 (IFI16), are key members of the hematopoietic interferon-inducible nuclear protein with a 200-amino-acid repeat (HIN-200) family. IFI204 serves as an intracellular DNA sensor that binds double-stranded DNA and participates in transcriptional regulation and innate immunity responses [ 14 , 15 ] . Its C-terminal HIN domains can specifically recognize both pathogen-derived and self-DNA. Upon DNA binding, IFI204 undergoes conformational changes and oligomerization, allowing its N-terminal pyrin domain to recruit the ASC adaptor and promote inflammasome assembly, ultimately leading to caspase-1 activation and pyroptosis [ 15 ] . In addition, IFI204-mediated DNA sensing functionally interacts with stimulator of interferon genes (STING) signaling, in which IFI204 acts as an upstream regulator of type I interferon production, thereby establishing a coordinated immune network [ 16 , 14 ] . Studies in myocardial infarction have demonstrated that IFI16/IFI204 promotes cardiomyocyte pyroptosis by sensing mtDNA, and that IFI204 knockdown alleviates cardiac damage in mice [ 17 ] . However, the role of IFI204 in regulating mtDNA-driven inflammation and pyroptosis in ischemic stroke remain poorly defined. Therefore, we propose that neuronal IFI204 serves as the central mediator that recognizes mtDNA and driving a vicious cycle involving pyroptosis, mitochondrial damage, and mtDNA release. This study aims to elucidate the functional interplay between the mtDNA–IFI204 axis and STING signaling in ischemic stroke. 2. Materials and Methods 2.1 Transgenic mice Neuron-specific IFI204 knockout (IFI204 cKO ) mice were generated by crossing Ifi204 floxed mice with NestinCre transgenic mice. The Ifi204 floxed allele, containing loxP sites flanking exons 2 to 4, was generated using CRISPR/Cas9 technology by GemPharmatech LLC (Jiangsu, China). The Nestin Cre mice, which express Cre recombinase under the control of the Nestin promoter on a C57BL/6J background, were obtained from Shanghai Model Organisms Center, Inc. Besides, wide-type (WT) C57BL/6J mice and and Sting1 knockout mice were purchased from GemPharmatech LLC (Jiangsu, China). All mice were housed under specific pathogen-free conditions in a controlled environment (20–22°C, 12-h light/dark cycle) with food and water ad libitum. Male and female mice aged 6 to 8 weeks were used for subsequent experiments. 2.2 Genotyping Mouse genotyping was performed by polymerase chain reaction (PCR) analysis of genomic DNA. Tail biopsies were collected, and DNA was extracted using a standard alkaline lysis method [ 18 ] . PCR amplification was conducted using a BIO-GENER GE4852T thermocycler (BIO‐GENER, China) with primer sets synthesized by Sangon Biotech. Primers specific for the Ifi204 flox , and Nestin Cre transgenes were listed in Supplementary Table 1. The resulting PCR products were separated on a 3% agarose gel and visualized under ultraviolet light after staining with SYBR Green. 2.3 MCAO Model Focal cerebral ischemia was induced via transient occlusion of the right middle cerebral artery (MCA) for 60 min, followed by reperfusion, as previously described [ 19 ] . Mice were anesthetized with isoflurane (1 ~ 1.5%) delivered in medical air. After a midline cervical incision, the right common carotid artery was temporarily ligated. A silicon-coated monofilament (Beijing Cinontech, China) was introduced through the external carotid artery and advanced into the internal carotid artery until occlusion of the right MCA. Successful occlusion and subsequent reperfusion were confirmed in each animal using Laser Speckle Doppler Flowmetry (PeriCam PSI Z; Perimed, Sweden). Only animals exhibiting a reduction in regional cerebral blood flow (rCBF) greater than 70% during occlusion were included in the study. Throughout the procedure, body temperature was maintained at 37.0 ± 1.0°C using a heating pad. All surgical procedures were performed by an experimenter blinded to the group allocations. 3.4 Measurement of Brain Infarct and Oedema Volume Cerebral infarct volume was quantified using 2,3,5-triphenyltetrazolium chloride (TTC; Sigma, USA) staining. Briefly, brain tissues were rapidly harvested and coronally sectioned into 6 slices with a thickness of 1 mm. The slices were incubated in 2% TTC solution at 37°C for 10 min in the dark, fixed with 4% paraformaldehyde (PFA), and subsequently photographed using Epson Perfection V19 Scanjet (Seiko Epson, Japan). The infarct area on each slice was measured using Image J software (National Institutes of Health, USA). The infarct volume was calculated as a percentage using the following formula: [(contralateral hemisphere volume − non-infarcted ipsilateral volume) / contralateral hemisphere volume × 2] × 100%. The extent of oedema in the ipsilateral hemisphere was calculated separately as: [(ipsilateral hemisphere volume − contralateral hemisphere volume) / (contralateral hemisphere volume × 2)] × 100%. 3.5 Neurobehavioral Tests Neurobehavioral assessments were performed before surgery and periodically up to 30 days after middle cerebral artery occlusion (MCAO) (Fig. 3 ). Sensorimotor function was evaluated using a battery of tests, including the adhesive removal test, the corner test, and the modified Neurological Severity Score (mNSS). Long-term learning and memory functions were assessed using the novel object recognition (NOR) test and the Morris water maze test, following established protocols. Testing was conducted in a controlled environment with consistent lighting and noise levels, and the experimenter was blinded to the group assignments. Modified Neurological Severity Score Test Neurological function was assessed using mNSS, a composite evaluation of motor, sensory, reflex, and balance functions. The mNSS scale ranges from 0 to 18, with 0 indicating normal neurological deficit and 18 indicating most severe neurological impairment. The assessment included tests of spontaneous activity, symmetry of limb movement, forepaw outstretching, climbing, body proprioception, and response to vibrissae touch [ 20 ] . Rotarod Test Motor coordination and balance were evaluated using an automated rotarod apparatus. Prior to surgery, all mice underwent a two-day training and baseline testing protocol. During each session, the rotational speed increased linearly from 4 to 40 revolutions per minute over a 5-min period. Mice that fell within 5 s of the start were immediately placed back on the rod for another attempt. Each mouse performed three trials per day with a minimum inter-trial interval of 10 min. The longest latency to fall from the three trials was recorded as the baseline performance. Postoperativel testing was conducted at designated time points using the same acceleration protocol. The apparatus was cleaned with 70% ethanol between animals. Adhesive Removal Test The Adhesive removal test was used to evaluate the sensitivity and dyspraxia of the forepaw [ 21 ] . Adhesive tapes measuring 3 × 3 mm 2 were attached to the hairless area of the right forepaw. The time required for each mouse to detect and remove the tapes were recorded. If a mouse failed to touch or remove the tapes within 120 s, the time was recorded as 120 s. Mice were trained once daily for three consecutive days before surgery, and the performance on the final training day was recorded as the baseline value. Novel Object Recognition Test The NOR test was conducted in an open-field arena between postoperative days 21 and 23. Mice were first habituated to the empty arena for 30 mins on two consecutive days. Twenty-four hours after the final habituation session, each mouse underwent a 5 min familiarization phase in which two identical objects were placed in symmetrical positions within the arena. After a 1 h interval, one familiar object was replaced with a novel object for a 5 min test phase. The arena and objects were cleaned with 75% ethanol between trials to eliminate olfactory cues. All sessions were video-recorded, and exploratory behavior, defined as directing the nose toward an object within a distance of approximately 2 cm, was analyzed using Smart v3.3.06 software (Panlab, Harvard Apparatus). The recognition index was calculated as the percentage of time spent exploring the novel object relative to the total exploration time for both objects. Morris water maze test Mice were subjected to Morris water maze testing beginning on day 24 after MCAO in a circular white pool with a diameter of 1.2 m and a height of 0.5 m, filled with water maintained at 22–25°C [ 22 ] . A hidden escape platform (10 cm diameter) was submerged 1 cm below the water surface in a predefined target quadrant. The test consisted of three phases. During the pre-training phase, the platform was made visible with an external cue, amd mice completed four 60 s trials. Mice that failed to locate the platform were gently guided to it. During the acquisition phase, the platform was hidden, and mice performed four 60 s trials per day for five consecutive days. Animals that did not locate the platform were guided to it and allowed to remain for 15 s to observe spatial cues. The daily mean escape latency and swimming path length was recorded. Finally, a probe trial was conducted on day 30 with the platform removed. Each mouse was allowed to swim freely for 60 s, and the time spent in the target quadrant, the number of crossings over the former platform location, and the overall swimming speed were recorded. 2.6 Virus Production and Stereotaxic Injection Recombinant adeno-associated viruses (AAVs) targeting neurons were used, including a control virus (rAAV-hSyn-MCS-3FLAG-EGFP, AAV-NC) and a virus overexpressing IFI204 (rAAV-hSyn-IFI204-3FLAG-EGFP, AAV-IFI204), both with titers of at least 2.00 × 10¹ 3 v.g./mL (GeneChem, Shanghai, China). Viral sequences are listed in Supplementary Table 2. A total volume of 5 µL of virus was stereotaxically injected into right lateral ventricle at the following coordinates: coordinates: anterior-posterior, − 0.2 mm; medial-lateral, − 1.0 mm; dorsal-ventral, − 2.2 mm from bregma. Following a three -week period to allow sufficient gene expression, transient focal cerebral ischemia was induced by MCAO. 2.7 Transmission Electron Microscopy Tissue specimens from the ipsilateral infarct border zone were sequentially fixed in 2.5% glutaraldehyde and 1% osmium tetroxide. After dehydration and embedding, ultrathin sections (50–60 nm) were prepared and imaged using a transmission electron microscope (JEM-1400; JEOL, Japan). Mitochondrial morphology was analyzed by quantifying mitochondrial number and measuring their length. Mitochondrial counts were performed manually, length measurements were conducted using Image J software. Mitochondria displaying ultrastructural damage, characterized by loss of cristae and/or disruption of mitochondrial membrane, were identified as damaged. 2.8 Cell Culture and Treatment The HT-22 immortalized mouse hippocampal neuronal cell line was obtained from Procell (Wuhan, China) and cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO₂. Oxygen-glucose deprivation and reperfusion (OGD/R) was induced as previously described [ 23 ] . Briefly, cells were rinsed with phosphate-buffered saline (PBS) and incubated in glucose-free DMEM (Gibco, USA). Subsequently, the cells were transferred to a sealed anaerobic chamber (Billups-Rothenberg), which was equilibrated with a gas mixture of 95% N₂ and 5% CO₂ to induce OGD for 6 hours. Control cells were maintained under normoxic conditions (95% air, 5% CO₂). After the OGD period, cells were returned to the normoxic incubator, and the medium was replaced with standard glucose-containing DMEM to initiate reperfusion. Neurons were treated with pharmacological agents immediately before and after OGD. Necrosulfonamide (NSA; MedChemExpress, USA) was dissolved in dimethyl sulfoxide (DMSO) and applied at a final concentration of 2 µM. Mitoquinone (MitoQ; MedChemExpress, USA) was dissolved in DMSO and applied at a final concentration of 1 µM. An equivalent volume of DMSO was administered to the vehicle control group. 2.9 Small RNA (siRNA) Interference HT-22 cells were cultured in antibiotic-free medium without penicillin and streptomycin until approximately 30% confluency was reached. IFI204 siRNA and the negative control siRNA were purchased from Sangon Biotech (Shanghai, China), and the corresponding sequences are listed in Supplementary Table 2. HT-22 cells were transfected with siRNA using GenMute™ siRNA Transfection Reagent (SignaGen Laboratories, USA) according to the manufacturer’s instructions 2.10 Cytosolic mtDNA Extraction and Quantification For mtDNA analysis, cells were divided into two equal aliquots. One aliquot was lysed in 300 µL of 50 mM NaOH by boiling at 95°C for 30 min to solubilize total DNA, followed by neutralization with 30 µL of 1 M Tris-HCl (pH 8.0). This whole-cell lysate was used as a normalization control for total mtDNA quantification. The second aliquot was permeabilized in approximately 300 µL of buffer containing 150 mM NaCl, 50 mM HEPES (pH 7.4), and 25 µg/mL digitonin (EMD Chemicals, USA) with rotation at room temperature for 10 min. The homogenate was then centrifuged three times at 980 g for 3 min at 4°C to pellet intact cells. The resulting supernatant (cytoplasmic fraction) was further clarified by centrifugation at 17,000 g for 20 min to remove residual debris. Quantitative PCR (qPCR) was performed on both the whole-cell lysate and the cytoplasmic fraction. Primers targeting nuclear DNA (Tert) and mtDNA (D-loop, Cox1, Cox3 and ND1) were used, with their sequences detailed in Supplementary Table 1. The mtDNA abundance (Ct value) obtained from the whole-cell lysate was used to normalize the mtDNA levels measured in the cytoplasmic fraction [ 24 ] . 2.11 scRNA-seq data processing The single-cell RNA sequencing dataset (GSE227651) was obtained from the Gene Expression Omnibus (GEO) database. Samples from the Sham and MCAO_D1 groups were selected for analysis. Raw sequencing reads were aligned to the mouse reference genome (mm10) and quantified using CellRanger software (version 7.1.0). Subsequent data processing and analysis were performed with the Seurat R package (version 4.3.0). Cells were subjected to standard quality control filtering, with exclusion criteria including fewer than 200 or more than 5,000 detected genes or a mitochondrial gene proportion exceeding 10%. Gene expression was normalized and variance-stabilized using the SCTransform method. To mitigate batch effects between samples, data integration was performed using the IntegrateData function. Dimensionality reduction was conducted using principal component analysis (PCA), followed by graph-based clustering. For visualization, t-distributed stochastic neighbor embedding (t-SNE) was applied using the top principal components via the RunTSNE function. Differential expression analysis between the Sham and MCAO_D1 groups was performed specifically within annotated neuronal clusters, using the FindMarkers function in Seurat. Identified differentially expressed genes (DEGs) were intersected with the MitoCarta3.0 database to obtain a neuron-enriched mitochondrial gene set. The expression pattern of these gene across samples was visualized in a heatmap. Gene Set Variation Analysis (GSVA) was applied to calculate single-cell enrichment scores for the gene set, and score distributions were compared between groups. Gene Set Enrichment Analysis (GSEA) was subsequently performed to evaluate coordinated regulation at the group level. 2.12 RNA sequencing Publicly available human stroke transcriptomic data (GSE162955) were obtained from the GEO database. GSEA was performed on this dataset (infarct core vs. contralateral tissue) using the clusterProfiler R package to test for the enrichment of the cytosolic DNA-sensing pathway in the infarct core. Total RNA was extracted using TRIzol reagent (Thermo Fisher, USA) and subjected to quality control, with inclusion criteria of RNA concentration greater than 50 ng/µL, RNA integrity number greater than 7.0, and total RNA amount exceeding 1 µg. Strand-specific sequencing libraries were prepared from poly(A)-selected mRNA. Libraries were sequenced on an Illumina NovaSeq™ 6000 platform using paired-end 150 bp reads. Raw sequencing data were processed with Cutadapt to remove adapters and low-quality bases. Clean reads were aligned to the mouse reference genome (Ensembl v112) using Hisat2. Transcript assembly and expression quantification (in FPKM) were performed using StringTie. Differential expression analysis was carried out using DESeq2 for datasets with biological replicates, with thresholds set at |log₂ (fold change)| ≥ 1.2 and an adjusted p-value (q-value) < 0.05. Downstream analyses included functional enrichment analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, GSEA, and protein-protein interaction network construction using STRING. All analyses were performed using the OmicStudio cloud platform. 2.13 Molecular docking The protein-protein interaction between IFI204 and STING was investigated through molecular docking. Amino acid sequences of mouse IFI204 and STING were retrieved from the UniProt database. The three-dimensional structure of the IFI204-STING complex was predicted using AlphaFold 3 in a fully automated mode. The top-ranked predicted complex model was subjected to energy minimization using the AMBER24 software package with the ff14SB force field. The binding affinity of the energy-minimized complex was then evaluated using the online PRODIGY tool. The interaction model exhibiting the most favorable binding energy was visualized and analyzed using PyMOL 2.5.3. In the resulting structural representation, IFI204 and STING are displayed as cartoon models in wheat and violet, respectively, with key interacting residues highlighted as sticks in the corresponding colors. 2.14 GST pull-down assay For the glutathione-S-transferase (GST) pull-down assay, DNA fragments encoding full-length or truncated mouse IFI204, including amino acids residues 1–88, 213–413, and 417–615, were cloned into the pET-GST vector to generate GST fusion constructs. These constructs were expressed in E. coli BL21. The resulting GST fusion proteins were affinity-purified using glutathione-Sepharose beads. For the binding assay, purified GST fusion proteins immobilized on beads were incubated with lysates derived from the cerebral ischemic penumbra for 4 h at 4°C with gentle agitation. After extensive washing, bound proteins were eluted and analyzed by Coomassie Brilliant Blue staining and immunoblotting. 2.15 Immunoblot For total protein extraction, cells or brain tissues were lysed using RIPA buffer. Cytoplasmic and mitochondrial proteins were isolated using a commercial Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China) and Mitochondrial Protein Extraction Kit (Proteintech, China) respectively, according to the manufacturer's instructions. Protein samples were separated by SDS-PAGE on 8 ~ 15% gels and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk prepared in Tris-buffered saline (TBS) for 1 hour at room temperature and then incubated with primary antibodies overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Immunoreactive bands were visualized using enhanced chemiluminescence reagent (Willget biotech, China) and detected with Azure 500 (Azure Biosystems, USA). Band intensities were quantified using ImageJ software. Details of the primary antibodies used are provided in Supplementary Table 3. 2.16 Immunostaining Immunofluorescence staining was performed on both brain tissue sections and cultured HT-22 cells. For tissue preparation, mice were deeply anesthetized and transcardially perfused with PBS, followed by 4% PFA. Brains were harvested, post-fixed in 4% PFA overnight at 4°C, cryoprotected in 30% sucrose until sinking, and sectioned into 20 µm coronal slices using a cryostat (Leica, Germany). HT-22 cells were grown on confocal dish. All samples were blocked for 1 hour at room temperature in PBS containing 10% donkey serum, 1% bovine serum albumin (BSA), and 0.1% Triton X-100. Samples were then incubated with primary antibodies (Supplemental Table 3) overnight at 4°C. After three washes with PBS, samples were incubated with appropriate fluorophore-conjugated secondary antibodies (Supplemental Table 3) for 2 hours at room temperature, followed by nuclear counterstaining with DAPI for 15 min. Tissue sections were mounted with Fluoromount (Sigma, USA). Images were acquired using a confocal microscope (Olympus FV3000, Japan), and fluorescence signals were quantified using ImageJ software. 2.17 FJC Staining Fluoro-Jade C (FJC) staining was used to identify degenerating neurons [ 25 ] . According to the instructions provided with the FJC ready-to-dilute staining kit (Biosensis, Australia), frozen brain sections mounted on gelatin-coated slides were dried at 50–60°C for 40 min, and immersed in 70% ethanol for 5 min, followed by three rinses in distilled water. Sections were oxidized in a 0.06% potassium permanganate solution for 10 min and then rinsed in distilled water for 1 min to terminate oxidation. Subsequently, sections were stained with FJC working solution (0.0004% in 0.1% acetic acid) for 10 min. Slides were washed three times with distilled water, air-dried at room temperature, and coverslipped with a non-aqueous mounting medium for imaging. 2.18 Detection and quantification of cytosolic DNA release Mitochondria in live HT-22 cells were labeled with 200 nM MitoTracker Deep Red (Beyotime, China). at 37°C for 30 min. After fixation and immunofluorescent staining for DNA and DAPI, cytosolic DNA foci were quantified by manually counting extranuclear DNA puncta located outside both nucleus and the perimeter of MitoTracker-labeled mitochondria [ 26 ] . Cells containing more than two such extramitochondrial DNA foci were considered positive for cytosolic DNA release. 2.19 Mitochondrial membrane potential Mitochondrial membrane potential was evaluated using the JC-1 fluorescent probe (Mitochondrial Membrane Potential Assay Kit, Beyotime, China). According to the manufacturer's protocol, cells were incubated with JC-1 working solution. In polarized mitochondria, JC-1 forms aggregates emitting red fluorescence, whereas mitochondrial depolarization leads to monomeric forms emitting green fluorescence. Fluorescence images of both red and green signals were acquired using a confocal microscope and analyzed with Image J software. The ratio of red to green fluorescence intensity was calculated as an index of mitochondrial membrane potential. 2.20 MitoSOX staining Mitochondrial superoxide levels in HT-22 cells were assessed using the MitoBright ROS Deep Red fluorescent probe (Dojindon, Japan). Cells were cultured on confocal dishes, and a 10 µmol/L working solution was prepared by diluting the DMSO stock solution in culture medium. After removal of the growth medium, cells were incubated with the working solution at 37°C for 30 min, and subsequently washed. Fluorescence images were acquired using a confocal microscope equipped with a 561 nm laser and a 640–700 nm emission filter. 2.21 Statistics Data are presented as mean ± standard deviation (SD) and analyzed using GraphPad Prism software (version 9.0, La Jolla, CA, USA). Normality of data distributions was verified via the Shapiro-Wilk test. For comparisons between two group, two-tailed unpaired Student’s t-test was applied for normally distributed data, whereas the Mann-Whitney U test was applied for non-normally distributed data. Survival analysis was performed using the log-rank (Mantel-Cox) test. Comparisons among multiple groups were conducted using one-way or two-way analysis of variance (ANOVA) followed by Šídák's multiple comparisons test for normally distributed data, or the Kruskal-Wallis test for non-normally distributed data. Repeated-measures data (e.g., behavioral tests) were analyzed using repeated-measures two-way ANOVA, followed by Tukey’s post hoc test for multiple comparisons as appropriate. p < 0.05 was considered statistically significant. 3. Results 3.1 Neuronal IFI204 is upregulated after acute brain ischemia To investigate the innate immune response following ischemic insult, we analyzed the publicly available human stroke transcriptomic dataset GSE162955. GSEA revealed that genes associated with cytosolic DNA-sensing pathway were significantly enriched in the infarct core (Fig. 1 A). We next employed a mouse model of MCAO to examine alterations in this pathway, where differential expression analysis identified Ifi204 as being significantly upregulated (Fig. 1 B, C). IFI204 protein expression was evaluated by immunoblotting of peri-infarct tissue and by immunostaining of coronal brain sections from sham-operated mice and MCAO mice at 6 h, 1d, 3d, 5d, and 7d after MCAO (Fig. 1 D–G). IFI204 expression exhibited dynamic changes over time following ischemic stroke. As Fig. 1 D depicts, IFI204 expression increased after MCAO, reaching a peak of approximately 3.1-fold at 1 d compared with the control, followed by a gradual decline. Immunofluorescence staining confirmed the western blot results, showing a marked increase in IFI204 expression in neurons located in the peri-infarct region at 1 d after MCAO (Fig. 1 F, G). In addition, IFI204 signal displayed minor colocalization with GFAP (astrocytes marker), Iba1 (microglia and monocyte-derived macrophages marker), or CD31 (endothelial cells marker) (Fig. S1 A and B). To further validate these findings in vitro, HT-22 cells were subjected to OGD/R, and immunoblot analysis was performed at 6, 12, and 24 h (Fig. 1 H). IFI204 protein was detectable under all conditions, with expression peaked 12 h after OGD/R and declining at 24 h (Fig. 1 H). Immunofluorescence analysis demonstrated that IFI204 was present in both the control and OGD/R-treated cells and was predominantly localized in the nucleus. However, following OGD/R, a portion of IFI204 signal was detected in the cytoplasm (Fig. 1 I), suggesting its potential role in cytoplasmic innate immune responses under ischemic conditions. 3.2 Neuron–specific IFI204 deletion attenuates ischemic stroke To further determine the role of neuronal IFI204 in ischemic injury, neuron-specific IFI204 cKO mice were generated by crossing IFI204 f/f mice with Nestin Cre mice (Fig. S2A and B). Immunofluorescence staining confirmed the selective deletion of IFI204 protein in neurons (NeuN + cells), with no observable change in non-neuronal colocalization (Fig. S2C). The decrease in IFI204 levels in the of IFI204 cKO mice was confirmed by immunoblot analysis (Fig. S2D). Infarct volume was examined by TTC staining 24 h after MCAO (Fig. 2 A and B). Successful induction of MCAO was verified by reduced blood flow during occlusion, as measured by laser Doppler flowmetry (Fig. S3A). To assess whether neuron-specific IFI204 deletion confers sex-dependent benefits, MCAO was also performed in female mice. Both male and female IFI204 cKO mice exhibited reduced infarct size and oedema volume compared with littermate controls (Fig. 2 C, and D; Fig. S3D). Neurodegeneration was evaluated by quantifying FJC + neurons in the cortex and striatum of the ipsilateral hemisphere 1 d after sham or MCAO surgery (Fig. 2 E). Sham-operated IFI204 f/f and IFI204 cKO mice displayed similarly low levels of FJC + cells. After MCAO, a marked increase in FJC + cells was observed in both regions. Notably, in the cortex, IFI204 f/f mice exhibited a significantly higher density of degenerating neurons than IFI204 cKO mice following ischemia. In contrast, striatal neuronal degeneration did not differ significantly between the two groups post‑MCAO (Fig. 2 F). These data indicate that neuronal IFI204 deletion partially attenuates ischemia‑induced neuronal death. 3.3 Deletion of neuronal IFI204 improves neurological function To evaluate neurological deficits after ischemic damage, a battery of behavioral tests was performed to comprehensively examine sensorimotor functions over a 30-day period after MCAO (Fig. 3 A). The mNSS, rotarod, and adhesive tape removal tests are applied to examine the sensorimotor functions of adult male mice. Mice subjected to MCAO showed pronounced sensorimotor impairments lasting at least 4 weeks. In contrast, neuron-specific IFI204 deletion significantly ameliorated sensorimotor dysfunction, as evidenced by lower mNSS scores, longer time to fall in the rotarod test, and shorter response time for adhesive removal from the contralateral forepaw (Fig. 3 B–D). Cognitive function was assessed using the NOR test at 21 d post-stroke. Both MCAO groups demonstrated impaired performance; however, the preference index was higher in the IFI204 f/f group than in the IFI204 cKO group (Fig. 3 E, F). Spatial learning and memory were further assessed using the Morris Water Maze (MWM) test (Fig. 3 G). During acquisition phase, IFI204 f/f mice exhibited progressive reductions in escape latency and path length, whereas IFI204 cKO mice displayed relatively shorter escape latency and swimming path length (Fig. 3 H, I). During the probe trial, IFI204 cKO mice spent more time in the target quadrant compared with IFI204 f/f mice, although the number of platform crossings did not differ significantly between groups (Fig. 3 J, K). Importantly, swimming speed showed no significant difference between the two MCAO groups (Fig. 3 L), indicating comparable visual and motor abilities in IFI204 f/f and IFI204 cKO mice during behavioral testing. 3.4 Neuron-specific IFI204 knockdown reduces pyroptosis and pyroptotic mitochondrial damage GSDMD is the critical executor of pyroptosis. Neuronal pyroptosis in the cerebrum, including the cortex and striatum, was assessed by double immunofluorescence staining for GSDMD and NeuN. IFI204 knockout efficiently abrogated GSDMD expression after MCAO, as indicated by a marked reduction in the number of GSDMD + neurons in cortex, whereas no significant difference was observed in striatum (Fig. 4 A). Consistent with these findings, IFI204 deletion also diminished the GSDMD signal colocalized with mitochondria (Fig. 4 B). Transmission electron microscopy revealed that MCAO induced plasma membrane disruptions and mitochondrial abnormalities, characterized by shortened, swollen mitochondria with disorganized cristae (Fig. 4 C). Compared with IFI204 f/f mice after MCAO, IFI204 cKO mice exhibited a partial attenuation of these structural injuries, as quantified by increased mean mitochondrial length and a reduced proportion of damaged mitochondria (Fig. 4 C). The number of mitochondria per cell did not differ significantly between the two groups (Fig. 4 C). Subsequently, mitochondrial and cytosolic fractions were analyzed by immunoblotting to assess the distribution of full-length GSDMD and GSDMD-N, as well as the release of mitochondrial components including cytochrome c (Cyto c; mitochondrial intermembrane space) and aconitase 2 (ACO2; mitochondrial matrix) (Fig. 4 D, E). Concurrently, we examined the expression of mitochondrial transcription factor A (TFAM), a key regulator of mtDNA stability and mitochondrial function [ 27 ] . Disruption mitochondrial integrity can lead to aberrant TFAM expression. Western blot analysis demonstrated that MCAO significantly increased the levels of GSDMD-N in both cytosolic and mitochondrial fractions, accompanied by cytosolic accumulation of Cyto c, ACO2, and TFAM compared to sham-operated controls. These coordinated alterations were markedly attenuated in IFI204 cKO mice subjected to MCAO, indicating that IFI204 deletion alleviated pyroptosis activation and mitochondrial membranes disruption after ischemic stroke (Fig. 4 D, E). scRNA-seq (GSE227651) data from animal models revealed a general downtrend among mitochondria-associated genes in neurons following MCAO, as determined by GSEA (Fig. S4A-D). This transcriptional response, involving genes related to mitochondrial quality control, the electron transport chain (ETC), and oxidative phosphorylation, likely reflects a mitochondrial stress response to acute ischemic injury. Given that altered mitochondrial membrane permeability compromises ETC electron transfer, we focused on two ETC components, ubiquinol-cytochrome c reductase core protein 1 (UQCRC1) and cytochrome c oxidase subunit 8A (COX8A). Western blot confirmed that COX8A was upregulated after MCAO, whereas this effect was suppressed by IFI204 knockout (Fig. 4 F), suggesting that attenuation of pyroptosis‑driven membrane damage mitigates the acute mitochondrial stress response. Consistent with the alleviated mitochondrial damage, oxidative DNA damage was reduced, as evidenced by a significant decrease in 8‑OHdG + neurons in IFI204‑deficient mice post‑MCAO (Fig. 4 G). 3.5 Loss of IFI204 in HT-22 triggers mtDNA release during pyroptosis via GSDMD pore formation In order to further validate the role of IFI204 during neuronal pyroptosis in vitro, IFI204 expression was silenced using siRNA (Fig. S5A). Knockdown efficiency was confirmed by qRT-PCR and immunoblotting (Fig. S5B, C). Immunoblot analysis of subcellular fractions showed that OGD/R increased the GSDMD-N levels in both cytosolic and mitochondrial fractions, an effect attenuated by IFI204 knockdown, suggesting that IFI204 promotes both GSDMD activation and its mitochondrial translocation (Fig. 5 A, B). This observation was further supported by a reduction in the co-localization of GSDMD-N with MitoTracker following IFI204 knockdown (Fig. 5 C). As mitochondrial GSDMD-N pores can dissipate membrane potential and trigger the release of mtDNA and mitochondrial reactive oxygen species (mtROS) [ 9 ] , we next assessed these downstream events. Cytosolic double-stranded DNA (dsDNA) was defined by the absence of co-localization with DAPI and MitoTracker signals (Fig. 5 D). OGD/R increased the number of cytoplasmic dsDNA foci per cell, accompanied by elevated mitochondrial superoxide levels, as detected by the mitoSOX probe, and a shift from red (J-aggregates) to green (monomers) fluorescence in JC-1 staining, indicating mitochondrial membrane depolarization. These alterations were partially reversed by IFI204 knockdown (Fig. 5 D-I). We hypothesized that the cytoplasmic dsDNA originated from leaked mtDNA. Co-immunostaining for mitochondrial markers TFAM, and dsDNA following OGD/R revealed mitochondrial origin of the cytosolic DNA foci (Fig. 5 J). Furthermore, qPCR analysis of isolated cytosolic DNA demonstrated an increased mtDNA copy number after OGD/R, which was reduced by IFI204 knockdown (Fig. 5 K). Collectively, these findings indicate that IFI204 regulates mtDNA leakage, likely by facilitating GSDMD pore formation on mitochondrial membranes. This conclusion is further supported by Western blot results showing that IFI204 knockdown preserved mitochondrial membrane integrity, as evidenced by reduced cytosolic leakage of mitochondrial components, including TFAM, Cyto c, and ACO2, and by suppression of respiratory chain components COX8A upregulation after OGD/R (Fig. 5 L, M). We next hypothesized that mtDNA released through GSDMD pores could amplify pyroptosis by activating the cytosolic DNA‑sensing pathway involving IFI204. Indeed, exogenous mtDNA stimulation after OGD/R further increased IFI204 expression, supporting the existence of a feedforward loop (Fig. 5 N). To delineate the role of GSDMD pores in mediating mtDNA release, we applied NSA, a specific inhibitor of GSDMD oligomerization. NSA treatment effectively suppressed pyroptosis, as indicated by reduced cleavage of GSDMD and caspase‑1 (Fig. S6A). Mitochondrial damage induced by OGD/R was partially attenuated, as reflected by restored TFAM expression and decreased GSDMD‑N levels in mitochondrial fractions (Fig. S6A-B). These effects were accompanied by a reduction in cytosolic mtDNA release after OGD/R (Fig. S6C-E). Furthermore, western blot and immunofluorescence analyses demonstrated that NSA treatment downregulated the expression of the cytosolic DNA sensor IFI204 (Fig. S6F, G). Together, these results support a self-amplifying loop in which mtDNA released via GSDMD pores activates IFI204, thereby further amplifing the pyroptotic cascade. To investigate whether attenuation of mitochondrial oxidative stress could prevent mtDNA leakage, we employed MitoQ, a mitochondrial-targeted antioxidant reported to protect mtDNA from oxidative damage and stabilizing TFAM [ 28 ] . In HT22 cells subjected to OGD/R, MitoQ treatment significantly reduced mitochondrial superoxide levels, as measured by mitoSOX staining, while exerting no effect on basal levels in control cells (Fig. S7A, C). Concurrently, MitoQ markedly decreased both the proportion of cells containing cytoplasmic dsDNA foci and the number of dsDNA foci per cell following OGD/R (Fig. S7B, D, E). These findings demonstrate that MitoQ protects against OGD/R-induced mtDNA leakage in neurons, likely by scavenging mtROS and preserving mtDNA integrity. In addition, MitoQ treatment suppressed the OGD/R-induced upregulation of IFI204 protein (Fig. S7F). 3.6 Re-expression of neuronal IFI204 aggravates neuronal pyroptosis and mitochondrial damage We next sought to determine whether supplementation of neuronal IFI204 could exacerbate mitochondrial damage and neuronal pyroptosis post-stroke. IFI204 was re-expressed in C57BL/6J IFI204 f/f and IFI204 cKO mice via stereotactic injection of an adeno-associated virus (AAV), with negative control (AAV-NC) treated mice serving as controls, three weeks prior to MCAO induction (Fig. 6 A). Under the hSyn promoter, the viruses with EGFP signals were predominantly observed in NeuN + neurons, and IFI204 expression was successfully restored by AAV9-hSyn-Ifi204 delivery (Fig. 6 B). Notably, neuronal IFI204 re-expression in MCAO mice promoted neuronal pyroptosis and partially reversed the protective phenotype. Specifically, the number of GSDMD + neurons in the peri-infarct cortex at 24 h after MCAO was substantially increased by elevated neuronal IFI204 levels in both IFI204 f/f and IFI204 cKO MCAO mice (Fig. 6 C), whereas no difference was detected in the striatum (Fig. S8A). Immunoblot and immunofluorescence co‑localization analyses collectively demonstrated that AAV-mediated IFI204 reconstitution increased GSDMD-N levels in both cytosolic and mitochondrial fractions and enhanced its co-localization with mitochondria markers (Tom20) (Fig. 6 D, H, I). Furthermore, IFI204 reconstitution also abolished the reduction in oxidative DNA damage in IFI204 cKO mice, with a concomitant elevation in the count of 8‑OHdG⁺ neurons, while no significant change was observed in IFI204 f/f mice (Fig. 6 E, Fig. S8B). As expected, ultrastructural analysis by electron microscopy revealed that transfection with AAV9-hSyn-Ifi204 aggravated abnormal mitochondrial morphology, leading to a reduction in mitochondrial length per neuron, and an increased proportion of damaged mitochondria characterized by swelling and disorganized cristae (Fig. 6 F, G). Stroke increased cytoplasmic expression of TFAM, Cyto c and ACO2, which was further exacerbated by IFI204 overexpression (Fig. 6 J). Meanwhile, the mitochondrial level of COX8A was amplified (Fig. 6 K). Collectively, the specific aggravation of injury-associated hallmarks upon IFI204 re-expression provides direct genetic evidence that IFI204 is a central driver of GSDMD-mediated mitochondrial dysfunction and neuronal pyroptosis after ischemic stroke. 3.7 IFI204 binding to STING via pyrin domain medicates neuronal pyroptosis and mitochondrial damage Transcriptomic analysis using RNA sequencing was performed to elucidate the underlying mechanisms and potential molecular targets through which IFI204 regulates neuronal pyroptosis under ischemic condition. A total of 1,150 DEGs were identified, including 227 upregulated and 923 downregulated genes, compared with IFI204 f/f MCAO group (Fig. 7 A). The key mediators Gsdmd and Sting1 were among the downregulated genes. GO enrichment analysis of biological processes revealed that the DEGs were predominantly enriched in immune-related terms, with "immune system process," "innate immune response," and "inflammatory response" ranking among the most significantly enriched terms (Fig. 7 B). GSEA further supported these observations by revealing significant positive enrichment of the "cytoplasmic pattern recognition receptor signaling pathway" and "pyroptosis" gene sets in control samples relative to IFI204 cKO MCAO mice (Fig. 7 C). Consistently, unsupervised heatmap analysis of DEGs within these gene sets showed a consistent downregulation pattern, prominently involving Gsdmd and Sting1 , in the IFI204 cKO group (Fig. 7 D). KEGG pathway analysis of the top enriched pathways highlighted the "NOD-like receptor signaling pathway" (Fig. 7 E). Subsequent GSEA confirmed this result and additionally demonstrated significant positive enrichment of the “cytosolic DNA-sensing pathway” in the IFI204 f/f group relative to IFI204 cKO group (Fig. 7 F). Heatmap visualization of these two key innate immune pathways revealed that Ifi204 , Gsdmd , and Sting1 clustered as core DEGs (Fig. 7 G). To further delineate functional interactome within the enriched pathway, a protein–protein interaction (PPI) network was constructed using DEGs annotated to the KEGG “NOD‑like receptor signaling pathway.” A high-confidence interaction between Ifi204 and Sting1 was identified, with a STRING confidence score greater than 900 (Fig. 7 H). Finally, Western blot analysis of STING protein expression in the same samples confirmed its significant downregulation in the IFI204 cKO group, thus validating the transcriptional changes at the protein level and linking them to the observed pyroptotic phenotype (Fig. 7 I). To validate the in vivo findings and further explore the cell-intrinsic mechanisms, we performed RNA sequencing on HT-22 neuronal cells subjected to OGD following transfection with either si-NC or si-IFI204. Differential expression analysis confirmed marked transcriptional reprogramming upon IFI204 knockdown (Fig. S9A). Analysis of the top 25 mitochondrial-associated GO terms revealed a pronounced enrichment of processes related to mitochondrial membrane integrity and dysfunction (Fig. S9B). Specifically, terms including “mitochondrial inner membrane,” “mitochondrial outer membrane,” “regulation of mitochondrial membrane potential,” “negative regulation of mitochondrial membrane permeability,” and “mitochondrial outer membrane permeabilization” were highly enriched. Critically, enrichment of term “positive regulation of cytochrome c release from mitochondria” provided direct transcriptional support for our biochemical observation that IFI204 deficiency reduces cytosolic Cyto c accumulation (Fig. 5 ), linking the transcriptomic changes to the suppression of mitochondrial permeabilization. KEGG pathway analysis identified the “NOD-like receptor signaling pathway” as one of the most significantly enriched pathways (Fig. S9C). GSEA further confirmed significant positive enrichment of both the “NOD-like receptor signaling pathway” and the “cytosolic DNA-sensing pathway” in control cells, consistent with observations in brain tissue (Fig. S9D). Sting1 is an important and core transcriptional co-activator in the NOD-like receptor signaling pathway (Fig. S9E). Finally, a PPI network constructed from genes within the NOD-like receptor signaling pathway again predicted a direct interaction between Ifi204 and Sting1, reinforcing this key molecular across both in vivo and in vitro models (Fig. S9F). The interaction between IFI204 and STING suggested by transcriptomic analyses was further examined at the protein level. In brain tissue from MCAO mice, immunofluorescence staining revealed co-localization of IFI204 and STING within NeuN + neurons (Fig. 7 J, K). Similarly, in OGD-treated HT-22 cells, IFI204 co-localized with STING in MAP2 + neuronal cells (Fig. 7 L, M). To define the structural domains responsible for IFI204–STING interaction, molecular docking analysis was performed, revealing a potential interaction between IFI204 and STING, with a predicted binding energy of -9.9 kcal/mol. The model indicated that residues within the N-terminal segment of IFI204, corresponding to its Pyrin domain (amino acids 1–88), formed hydrogen bonds with STING (Fig. 9 N). To test this prediction biochemically, a GST pull-down assay was conducted. Recombinant, purified GST-tagged full-length IFI204 successfully pulled down endogenous STING from post-MCAO brain lysates. This interaction was specifically mediated by the Pyrin domain of IFI204, as a GST-tagged Pyrin domain construct also bound STING, whereas GST-tagged HIN-A or HIN-B domains did not (Fig. 7 O). 3.8 Genetic ablation of STING partially rescues neuronal damage exacerbated by IFI204 overexpression Given the upstream regulation of IFI204 on STING in neurons, AAV-IFI204 was stereotactically delivered into the lateral ventricle of WT and Sting1-knockout (STING KO) mice, followed by MCAO induction (Fig. 8 A). Successful ablation of STING was confirmed by Western blot (Fig. 8 G). Immunofluorescence analysis of the cortex and striatum revealed a STING-dependent effect of IFI204 on neuronal pyroptosis. In the cortex, IFI204 overexpression significantly increased the number of GSDMD + neurons in WT mice, whereas this effect was partially attenuated in STING KO mice. In contrast, no significant differences were observed in GSDMD + neuron counts in the striatum among the groups (Fig. 8 B). Immunofluorescence analysis further showed that the co‑localization of GSDMD‑N with the mitochondrial marker Tom20 was greatly enhanced in WT mice overexpressing IFI204, whereas this enhancement was substantially attenuated in STING KO mice (Fig. 8 C). Besides, ultrastructural analysis by transmission electron microscopy indicated that cortical neurons from IFI204-overexpressing WT mice exhibited membrane disruption, reduced mitochondrial number and average length, and an increased proportion of damaged mitochondria. These pathological alterations were ameliorated in STING KO neurons (Fig. 8 D). Similarly, IFI204 overexpression led to a marked increase in the number of neurons exhibiting oxidative DNA damage, defined as 8-OHdG and NeuN double-positive cells in WT mice, which was partially mitigated in STING KO mice (Fig. 8 E). Reduced protein expression of molecules associated with pyroptosis and mitochondrial damage was also detected in STING KO mice receiving AAV-IFI204 (Fig. 8 F-I). These findings indicate that STING is required for IFI204-induced pyroptotic mitochondrial damage. 4. Discussion In the current study, we demonstrated that IFI204 deficiency attenuates neuroinflammation following MCAO. Genetic ablation of IFI204 reduced neuronal pyroptosis, alleviated mitochondrial damage and mtDNA leakage, and ultimately improved neurological outcomes (Fig. 9 ). Mechanistically, we identified a direct interaction between IFI204 and STING, mediated by the PYD of IFI204, which facilitates the membrane translocation and pore-forming activity of GSDMD, a key executioner of pyroptosis. The GSDMD pores subsequently formed on mitochondrial membranes serve as the direct conduit for mtDNA release into the cytosol. The leaked mtDNA further amplifies this cascade by activating the IFI204-STING axis, thereby establishing a self-sustaining inflammatory loop. Deletion of IFI204 disrupts this loop by suppressing STING-driven signaling, ultimately protecting ischemic neurons. This study identifies the p200 family protein IFI204 as a crucial DNA sensor linking hypoxic or ischemic injury to neuroinflammation. We demonstrate that IFI204 expression was upregulated in neurons following ischemic stroke, likely triggered by fragmented DNA released from damaged tissue. Another important finding is the subcellular redistribution of IFI204, which is predominantly nuclear under physiological conditions but partially translocated to the cytoplasm post-OGD. This redistribution appears to be functionally relevant, reflecting a shift from a nuclear DNA surveillance role to a cytoplasmic DNA-sensing function [ 29 ] . Regulation of this nucleo-cytoplasmic shuttling involves specific post-translational modifications, including nuclear localization signal (NLS) deacetylation-mediated nuclear import and chromosome region maintenance 1 (CRM1)-dependent nuclear export [ 30 – 32 ] . Importantly, subcellular localization influences downstream signaling outcomes. Nuclear IFI204 or IFI16 is associated with interferon-beta production, while cytoplasmic accumulation preferentially promotes inflammasome assembly [ 33 – 35 ] . Our observation of cytoplasmic IFI204 linked it to pyroptosis is consistent with its established role in activating the ASC/Caspase-1 inflammasome. The functional versatility of IFI204 is reflected in its mechanistic diversity in DNA recognition. IFI204 directly senses dsDNA in a non-sequence-specific manner via electrostatic interactions mediated by its C-terminal HIN domains [ 36 , 37 ] . This direct sensing is further enhanced by the cooperative assembly of DNA-bound complexes into higher-order filaments, driven by the N-terminal PYD domain [ 38 ] . In addition to direct recognition, IFI204 also engages in indirect DNA sensing within the nucleus as part of the DNA Damage Response (DDR) pathway, detecting genotoxic stress through protein–protein interactions with factors such as breast cancer type 1 susceptibility protein (BRCA1) and histone H2B [ 39 , 40 ] . Building on its DNA-sensing capability, IFI204 or IFI16 orchestrates pyroptosis through a complex network of synergistic pathways. IFI204 functions as a core inflammasomes component and directly nucleates its assembly. Inflammasome formation is further amplified through cooperative interactions with other innate immune molecules. For example, in a myocardial infarction model, IFI204 or IFI16 enhances inflammasome assembly efficiency by interacting with galectin-3, thereby amplifying the pyroptotic response [ 17 ] . Similarly, IFI204 acts through STING to drive pyroptosis during traumatic brain injury [ 41 ] , consistent with our observations in neurons. Beyond inflammasome assembly, IFI204 has been shown to interact specifically with TLR4 via the PYD region, facilitating its dimerization and enhancing nuclear factor kappa B (NF-κB) signaling, which leads to increased expression of essential pyroptosis components such as pro-IL-1β [ 42 ] . Moreover, IFI204 induces mitochondrial disorders, promoting ROS accumulation and exacerbating pyroptotic responses [ 41 ] . Mitochondrial dysfunction also intensifies inflammation by releasing mtDNA, which is sensed with high affinity by the IFI16 rs6940 variant, thereby activating the IFI16–caspase-1 inflammasome [ 43 ] . Notably, IFI204 or IFI16 directly interfaces with the cGAS-STING signaling pathway. IFI16 has been shown to recruit cGAS and enhance cGAMP synthesis, thereby facilitating STING activation [ 44 ] , or to function as a cGAS cofactor through its dsDNA-binding capacity [ 45 ] . Alternatively, STING recruitment by IFI16 or IFI204 activates downstream TBK1, leading to phosphorylation of IRF3 and NF-κB [ 14 , 46 ] . Depletion of IFI16 under dsDNA or 2'3'-cGAMP stimulation impairs STING phosphorylation [ 44 ] . Our RNA sequencing analysis revealed that IFI204 modulates STING signaling in neurons. Genetic knockout of STING suppressed its expression and partially attenuated IFI204 overexpression-induced neuronal pyroptosis. GST pull-down assays confirmed direct binding between IFI204 and STING, localizing the interaction to the PYD of IFI204. Consistent with our findings, previous co-immunoprecipitation studies in HSV-1-infected or DNA-stimulated human monocytes have demonstrated the PYD of IFI16 mediates its interaction with STING [ 44 , 47 ] . Mutations within this domain disrupted IFI16-STING complex formation in 293T cells [ 47 ] , suggesting that further identification of critical binding residues within the PYD of IFI204 is warranted. However, such studies are limited in their ability to exclude indirect interaction. Recent work in a traumatic brain injury model showed that SUMO-specific protease 7 (SENP7) directly interacts with IFI204 and catalyzes its deSUMOylation [ 41 ] . This modification enhances STING activation and strengthens IFI204–STING binding. In our earlier study, STING-mediated microglial pyroptosis involved the NLRP3 inflammasome pathway [ 22 ] . Therefore, the IFI204-STING axis in neurons may not function in isolation, suggesting potential cross-talk between IFI204-inflammasome and NLRP3 inflammasome that warrants further investigation. Our data identify GSDMD as a key molecular bridge linking pyroptotic signaling to mitochondrial impairment in neurons. Recent research have shown that gasdermin proteins target mitochondrial membranes through specific lipid interactions, with the N-terminal domain of GSDMD exhibiting high-affinity binding to cardiolipin and oligomerizing into 10–18 nm pores [ 48 ] . Disruption of both inner and outer mitochondrial membranes results in mitochondrial dysfunction, increased ROS production, and the release of mtDNA and cytotoxic proteins, thereby establishing mitochondrial damage as a critical irreversible step in pyroptosis execution [ 9 ] . This mechanism was validated in our neuronal model, in which treatment with the GSDMD oligomerization inhibitor NSA significantly reduced cytosolic mtDNA release following OGD, confirming GSDMD pore formation as a central event in neuronal mitochondrial damage during pyroptosis. Similarly, GSDME-NT, generated by caspase-3 cleavage, also targets mitochondria via cardiolipin binding, promoting mtDNA and cytochrome c release [ 49 ] . mtDNA leakage thus serves as a critical pathological link between mitochondrial damage and sterile inflammation, a process that is further amplified by downstream DNA sensors such as IFI204 and STING. The release process requires mitochondrial outer membrane permeabilization, which can be initiated through several distinct mechanisms under stress conditions. Some mechanisms depend on the formation of functional membrane pore structures. For example, voltage-dependent anion channel 1 (VDAC1) oligomerization disrupts mitochondrial membrane integrity and promotes mtDNA release [ 50 , 24 ] . Alternatively, pathological opening of the mitochondrial permeability transition pore (MPTP) can cause collapse of mitochondrial membrane potential and increase outer membrane permeability, thereby providing a physical channel for mtDNA escape [ 51 ] . During apoptosis, mitochondrial outer membrane permeabilization occurs through macropore formation in a BAX/BAK dependent manner, allowing herniation of the inner mitochondrial membrane into the cytoplasm and subsequent mtDNA release [ 52 ] . In parallel, studies have shown that during necroptosis, phosphorylated mixed lineage kinase domain-like protein (MLKL) translocates to and permeabilizes mitochondrial membranes, promoting mtDNA release in a microtubule-dependent manner [ 53 ] . Other mechanisms do not rely on direct pore formation but instead involve mitochondrial structural remodeling, abnormal mtDNA packaging or vesicle-mediated transport. For instance, abnormal mitochondrial fission induced by dynamin-related protein 1 (DRP1), accompanied by excessive mitochondrial ROS explosion, compromises DNA integrity and membrane permeability, indirectly facilitating mtDNA release [ 54 ] . Furthermore, deficiency in TFAM disrupts normal mtDNA packaging and stability, resulting in abnormal mtDNA copy number and cytoplasmic accumulation. Notably, the mitochondrial E3 ligase mitochondrial-anchored protein ligase (MAPL) initiates a unique pathway by packaging mtDNA into mitochondrial-derived vesicles [ 55 ] . These vesicles are transported to lysosomes where GSDME pore formation triggers lysosomal rupture, ultimately releasing mtDNA through a mechanism that bypasses direct mitochondrial membrane perforation. 5. Conclusions Overall, this study delineates a novel inflammatory cascade involved in ischemic neuronal injury. Hypoxic stress induces upregulation of IFI204, which directly interacts with STING, promoting inflammasome assembly, and leading to caspase-1 activation and GSDMD cleavage. The resulting GSDMD-NT forms pores on mitochondria membranes, facilitating mtDNA release. This mtDNA release further amplifies activation of the IFI204-STING-GSDMD axis, creating a feed-forward loop of inflammation and pyroptosis. These findings identify neuronal IFI204 as a central node integrating DNA sensing, innate immune signaling, and mitochondrial damage, and suggest potential therapeutic targets for mitigating neuroinflammation in ischemic stroke. Abbreviations AAV Adeno-associated virus ACO2 Aconitase 2 BRCA1 Breast cancer type 1 susceptibility protein BSA Bovine serum albumin COX8A Cytochrome c oxidase subunit 8A Cyto c Cytochrome c DAMP Damage-associated molecular pattern DEG Differentially expressed gene DMEM Dulbecco’s Modified Eagle Medium DMSO Dimethyl sulfoxide DRP1 Dynamin-related protein 1 dsDNA Double-stranded DNA ETC Electron transport chain FJC Fluoro-Jade C GO Gene Ontology GSEA Gene Set Enrichment Analysis GST Glutathione-S-transferase GSDMD Gasdermin D GSDMD-N Gasdermin D N-terminal fragment GSVA Gene Set Variation Analysis HIN Hematopoietic interferon-inducible nuclear protein HRP Horseradish peroxidase IFI16 Interferon gamma-inducible protein 16 IFI204 Interferon activated gene 204 IL-1β Interleukin 1 beta IL-18 Interleukin 18 KEGG Kyoto Encyclopedia of Genes and Genomes MAPL Mitochondrial-anchored protein ligase MCA Middle cerebral artery MCAO Middle cerebral artery occlusion MLKL Mixed lineage kinase domain-like protein mNSS Modified Neurological Severity Score MWM Morris water maze MitoQ Mitoquinone MPTP Mitochondrial permeability transition pore mtDNA Mitochondrial DNA mtROS Mitochondrial reactive oxygen species NF-κB Nuclear factor kappa B NLR NOD-like receptor NSA Necrosulfonamide NOR Novel object recognition OGD/R Oxygen-glucose deprivation and reperfusion PBS Phosphate-buffered saline PCA Principal component analysis PFA Paraformaldehyde PPI Protein-protein interaction PRR Pattern recognition receptor rCBF Regional cerebral blood flow ROS Reactive oxygen species scRNA-seq Single-cell RNA sequencing SD Standard deviation siRNA Small interfering RNA STING Stimulator of interferon genes TLR Toll-like receptor TFAM Mitochondrial transcription factor A TTC 2,3,5-Triphenyltetrazolium chloride UQCRC1 Ubiquinol-cytochrome c reductase core protein 1 VDAC1 Voltage-dependent anion channel 1 WT Wild-type Declarations Ethics approval and consent to participate All experiments involving animals were conducted according to the ethical policies and procedures approved by the ethics committee of the First Affiliated Hospital of University of Science and Technology of China (2025-N (A)-279). Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare no competing interests. Funding This work was supported by the National Natural Science Foundation of China (Nos. 82471310, 82471311, 82101368), the Anhui Provincial Natural Science Foundation (No. 2408085MH214). Authors' contributions PFX and NS: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing-Original Draft, Visualization. TQ and MYZ: Investigation, Methodology. RL, CRT and YYZ: Investigation. WH and PFX: Investigation, Resources, Writing-Review & Editing, Supervision, Project administration, Funding acquisition. All authors read and approved the final manuscript. Acknowledgements Not applicable References CHENG Z, WANG H R, GENG X K, et al. Time and Tissue Windows in Futile Reperfusion after Ischemic Stroke[J]. Aging Dis. 2025;16(5):2544–52. 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Supplementary Files Supplementarymaterials.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 23 Feb, 2026 Reviews received at journal 23 Feb, 2026 Reviews received at journal 15 Feb, 2026 Reviewers agreed at journal 01 Feb, 2026 Reviewers agreed at journal 21 Jan, 2026 Reviewers invited by journal 21 Jan, 2026 Editor assigned by journal 21 Jan, 2026 Submission checks completed at journal 20 Jan, 2026 First submitted to journal 19 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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07:27:14","extension":"html","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":187598,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8641308/v1/78d7881e0bce9b0b0a274b30.html"},{"id":100954497,"identity":"8f3ab59e-ab7a-4a91-9f92-d61cfd21b264","added_by":"auto","created_at":"2026-01-23 07:25:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":875510,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIFI204 expression is dramatically elevated in neurons after stroke\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) GSEA of differentially expressed genes in the cytosolic DNA-sensing pathway based on the transcriptomic data (GSE162955) of human brain after ischemia. n = 6.\u003c/p\u003e\n\u003cp\u003e(B) GSEA revealed significant positive enrichment (upregulation) of the cytosolic DNA-sensing pathway in MCAO samples. n = 5.\u003c/p\u003e\n\u003cp\u003e(C) Heatmap of representative genes belonging to the cytosolic DNA-sensing pathway in the ipsilateral hemisphere of sham and MCAO mice. n = 5\u003c/p\u003e\n\u003cp\u003e(D) IFI204 expression at the indicated time points following ischemic onset. n = 3.\u003c/p\u003e\n\u003cp\u003e(E) A representative image of NeuN immunofluorescence illustrates the infarct and peri-infarct region in the ipsilateral brain hemisphere (Scale bar = 200 μm). Double labeling of NeuN and IFI204 in peri-infarct area at 1d after MCAO (Scale bar = 50 μm).\u003c/p\u003e\n\u003cp\u003e(F) Enlarged area of NeuN and IFI204 double staining in the peri-infarct region at indicated time points after MCAO (Scale bar = 50 μm). n = 5.\u003c/p\u003e\n\u003cp\u003e(G) Quantification of the percentage of IFI204\u003csup\u003e+\u003c/sup\u003e neurons over total neurons. n = 5.\u003c/p\u003e\n\u003cp\u003e(H) Immunoblots of IFI204 in extracts of OGD-treated HT-22 cells (left) and quantification of IFI204 levels (right). n = 4.\u003c/p\u003e\n\u003cp\u003e(I) Immunocytochemistry for IFI204 and MAP2 after exposure to OGD/R for 12 h (Scale bar = 5 μm). n = 3.\u003c/p\u003e\n\u003cp\u003eData indicate means ± SD. *p \u0026lt; 0.05, ***p \u0026lt; 0.001, \u003csup\u003e###\u003c/sup\u003ep \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8641308/v1/8d04b1fcb0ea417e4aa76d43.png"},{"id":100954382,"identity":"a33962a7-ece0-4f54-9f92-f8b4f274952c","added_by":"auto","created_at":"2026-01-23 07:25:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2055311,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeuronal IFI204 silencing protects against ischemia-induced brain damage in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Scheme of timepoints for MCAO surgery.\u003c/p\u003e\n\u003cp\u003e(B) TTC staining for mouse brain after 1 day of MCAO or sham surgery.\u003c/p\u003e\n\u003cp\u003e(C, D) Statistical analysis of the infarct and edema ratio (% contralateral hemisphere).\u003c/p\u003e\n\u003cp\u003e(E, F) Representative images from peri-infart region stained with Fluoro-Jade C (FJC, green), and insets show a higher magnification view. (Scale bar = 50 μm). Analysis of the number of FJC\u003csup\u003e+\u003c/sup\u003e cells as a ratio of total cells.\u003c/p\u003e\n\u003cp\u003en = 5. Data indicate means ± SD. ns, not significant. *p \u0026lt; 0.05, **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8641308/v1/3c3276e9d2742aca352eae3a.png"},{"id":100954279,"identity":"942be7e0-602e-41d2-89f2-0947dde624be","added_by":"auto","created_at":"2026-01-23 07:24:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1275053,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIFI204\u003c/strong\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003ecKO\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e ameliorates long-term stroke outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Time line of MCAO surgery and behavioral tests.\u003c/p\u003e\n\u003cp\u003e(B-D) Sensorimotor deficits were evaluated before (base) and up to 28d after MCAO or Sham by the mNSS score, rotarod test and adhesive removal test.\u003c/p\u003e\n\u003cp\u003e(E, F) The representative movement tracks plots on the novel object recognition test. Recognition index analysis was performed.\u003c/p\u003e\n\u003cp\u003e(G) Representative swimming paths during the memory phase of the Morris water maze test.\u003c/p\u003e\n\u003cp\u003e(H, I) Spatial learning was assessed by the escape latency and path length (25-29 d after MCAO).\u003c/p\u003e\n\u003cp\u003e(J) The percentage of time spent in the target quadrant during the probe trial.\u003c/p\u003e\n\u003cp\u003e(K) Number of platform crossings during probe trials.\u003c/p\u003e\n\u003cp\u003e(L) Quantitation of the swim speed of the mice in the Morris water maze task.\u003c/p\u003e\n\u003cp\u003en = 10. Data indicate means ± SD. ns, not significant. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8641308/v1/ed98b7ea814c70061c76930e.png"},{"id":100954494,"identity":"8f1f2b62-af0c-4d87-a516-c9b6e0b55905","added_by":"auto","created_at":"2026-01-23 07:25:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1173196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIFI204 deficiency causes attenuated GSDMD-induced pyroptosis and mitochondrial damage in neurons.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative images of double immunofluorescent staining for NeuN and GSDMD in post-ischemic brains after MCAO (left, Scale bar = 50 μm). GSDMD positive neurons were quantitative analysis in cortex and striatum (right).\u003c/p\u003e\n\u003cp\u003e(B) Double-immunofluorescence staining for GSDMD (red) and Tom20 (green) in ischemic brains (left, Scale bar = 20 μm). Statistic of mitochondria colocalized with GSDMD-N (right).\u003c/p\u003e\n\u003cp\u003e(C) Representative transmission electron micrographs of neurons in the peri-infarct area. Rightmost panel showed the inset of the left panel at higher magnification. The yellow arrows showed pyroptosis pores on the plasma membrane (left). Mitochondrial (mito) mean length and number, and percentage of damaged mitochondria were quantified (right).\u003c/p\u003e\n\u003cp\u003e(D) Immunoblots probed for GSDMD-N in cytosolic and mitochondrial fractions.\u003c/p\u003e\n\u003cp\u003e(E) Cytoplasmic TFAM, ACO2 and Cyto c levels were analyzed by Western blot.\u003c/p\u003e\n\u003cp\u003e(F) Western blot analysis of mitochondrial extracts for UQCRC1 and COX8A.\u003c/p\u003e\n\u003cp\u003e(I) Representative immunofluorescence images of neurons stained with 8-OHdG and quantitative analysis of 8-OHdG\u003csup\u003e+\u003c/sup\u003e/NeuN\u003csup\u003e+\u003c/sup\u003e cells in the peri-infarct region (Scale bar = 50 μm).\u003c/p\u003e\n\u003cp\u003en = 5. Data indicate means ± SD. ns, not significant. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8641308/v1/65cbfb27888d42dc17459acf.png"},{"id":100954284,"identity":"d2a24253-7268-42c8-af6a-e23eec1f88de","added_by":"auto","created_at":"2026-01-23 07:24:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2219501,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of si-IFI204 on the pyroptosis of HT-22 cells induced by OGD/R.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B)HT-22 cells were transfected with Negative control-siRNA or IFI204-siRNA for 48 hours and underwent OGD (3 hours) /R (6 hours). Immunoblots probed for GSDMD and GSDMD-N in cytosolic and mitochondrial fractions.\u003c/p\u003e\n\u003cp\u003e(C) Confocal microscopy imaging stained for MitoTracker and GSDMD-N. Higher magnification images and 3D rendering of the boxed area are shown (left, Scale bar = 10 μm). Quantification of relative GSDMD-N fluorescence dye intensity in mitochondria (right).\u003c/p\u003e\n\u003cp\u003e(D) Representative confocal images of mitochondrial morphology (MitoTracker) and DNA foci (dsDNA) in HT-22 cells. Yellow arrowheads indicate cytosolic DNA foci (Scale bar = 10 μm).\u003c/p\u003e\n\u003cp\u003e(E) Immunofluorescence staining shows mitochondrial structure (MitoTracker and mitochondrial ROS levels (MitoSOX) under the indicated conditions. Composite images include bright field (Scale bar = 10 μm).\u003c/p\u003e\n\u003cp\u003e(F-H) Percentage of HT-22 cells showing cytosolic DNA foci,number of cytosolic DNA foci per cellfrom (D), and normalized mitoSOX fluorescence intensity were quantitative analysis.\u003c/p\u003e\n\u003cp\u003e(I) Quantification of mitochondrial potential by JC-1 aggregates (red) /JC-1 monomers (green) (scale bar = 10 µm).\u003c/p\u003e\n\u003cp\u003e(J) Representative microscopy image of dsDNA (red), TFAM (green) and mitochondria (MitoTracker, white) in HT-22 cells treated with OGD/R (Scale bar = 10 μm).\u003c/p\u003e\n\u003cp\u003e(K) Quantification of mtDNA copy number by PCR using ND1, D-loop, COX1, COX3 probe, from isolated cytosolic fractions of HT-22 cells.\u003c/p\u003e\n\u003cp\u003e(L) Cytoplasmic TFAM, ACO2 and Cyto c levels as analyzed by Western blot.\u003c/p\u003e\n\u003cp\u003e(M) Western blot analysis of mitochondrial extracts for UQCRC1 and COX8A.\u003c/p\u003e\n\u003cp\u003e(N) Treatment of HT-22 cells with exogenous mouse mtDNA enhanced IFI204 expression under both normal and OGD/R conditions.\u003c/p\u003e\n\u003cp\u003en = 5. Data indicate means ± SD. ns, not significant. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8641308/v1/2504f6d76a24b57ab471eb3c.png"},{"id":100954281,"identity":"2e4b11dc-925b-4b60-9ca2-43e14c39dafc","added_by":"auto","created_at":"2026-01-23 07:24:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1148704,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpecific enhanced expression of IFI204 in Neurons aggravates Pyroptosis and Mitochondrial Injury after MCAO\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagrams showing the vector construction: AAV9-hSyn-Ifi204-EGFP. 5 μL AAV particles\u0026nbsp;(2×10^13 v.g./ml)\u0026nbsp;was injected into the lateral ventricle 3 weeks before MCAO.\u003c/p\u003e\n\u003cp\u003e(B) Ifi204-expressing AAV was delivered to both IFI204\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eand IFI204\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e mice. Representative immunostaining images of the transfection of EGFP (green) reporter AAV into neurons (NeuN, purple) with enforced IFI204 (red) expression in brain (Scale bar = 20 μm).\u003c/p\u003e\n\u003cp\u003e(C) Representative pictures and quantification of double immunofluorescence staining of GSDMD (red) colocalized with NeuN (cyan) in cortex (Scale bar = 50 μm).\u003c/p\u003e\n\u003cp\u003e(D) Merged images represent overlays of TOM20 (cyan), GSDMD (red), and the nucleus (stained by DAPI, blue) (left, Scale bar = 20 μm). Statistic of mitochondria colocalized with GSDMD-N (right).\u003c/p\u003e\n\u003cp\u003e(E) Representative photomicrographs and quantification of 8-OHdG (red)/ NeuN (cyan) immunofluorescence staining in cortex (Scale bar = 50 μm).\u003c/p\u003e\n\u003cp\u003e(F, G) Representative transmission electron microscopy images of neuron in the peri-infarct area. The yellow arrows showed pyroptosis pores on the plasma membrane. Mitochondria number, mitochondrial mean length, and damaged mitochondria were analyzed.\u003c/p\u003e\n\u003cp\u003e(H, I) Immunoblots probed for GSDMD-N in cytosolic and mitochondrial fractions.\u003c/p\u003e\n\u003cp\u003e(J) TFAM, ACO2 and Cyto c cytoplasmic levels were quantified by Western blot analysis.\u003c/p\u003e\n\u003cp\u003e(K) Mitochondrial extracts were prepared and analyzed for UQCRC1 and COX8A.\u003c/p\u003e\n\u003cp\u003en = 5. Data indicate means ± SD. ns, not significant. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8641308/v1/e71176dc6612dfa1e973ce64.png"},{"id":100954375,"identity":"44f73b52-feee-47eb-9dca-07ebe7adfcd7","added_by":"auto","created_at":"2026-01-23 07:25:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3016030,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIFI204 medicates neuronal pyroptosis and mitochondrial damage through binding STING.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) DEGs between IFI204\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e versus IFI204\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e mice under ischemia condition analyzed by RNA sequencing. Volcano plot depicting the magnitude of gene expression change versus statistical significance. The numbers of down and up genes are indicated and colored blue and red, respectively. Thresholds for significance were set at adjusted. n = 3/4\u003c/p\u003e\n\u003cp\u003e(B) GO enrichment analysis of DEGs associated with biological process. Key terms related to Immunity and Inflammation are highlighted.\u003c/p\u003e\n\u003cp\u003e(C) GSEA plots. Enrichment scores (ES) are plotted against the ranked gene list. The running ES is shown as a red line; vertical black lines indicate gene positions within the gene set. Positive ES indicates upregulation in the IFI204\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e group.\u003c/p\u003e\n\u003cp\u003e(D) Heatmap of DEGs in cytoplasmic pattern recognition receptor signaling pathway and pyroptosis.\u003c/p\u003e\n\u003cp\u003e(E) KEGG pathway enrichment analyses of DEGs. Dot size represents the number of genes enriched in the pathway; color intensity corresponds to the statistical significance; the gene ratio (x-axis) is the percentage of significant genes over the total genes in a given pathway.\u003c/p\u003e\n\u003cp\u003e(F) GSEA enrichment plots for differentially expressed pathways of interest.\u003c/p\u003e\n\u003cp\u003e(G) Heatmap of DEGs in NOD-like receptor signaling pathway and cytosolic DNA-sensing pathway.\u003c/p\u003e\n\u003cp\u003e(H) Protein-protein interaction (PPI) network constructed for inflammation-associated genes in NOD-like receptor signaling pathway.\u003c/p\u003e\n\u003cp\u003e(I) Verification of RNA sequencing gene expression results was performed on Sting1 using western blot. n = 3 / 4.\u003c/p\u003e\n\u003cp\u003e(J-M) Intracellular localization of IFI204 (red) and STING (green) assessed by immunostaining in neurons. n = 3\u003c/p\u003e\n\u003cp\u003e(N) Binding model depicting the interaction between IFI204 and STING of mouse. IFI204 is colored in gold, and STING is colored in violet, with their binding sites shown as stick structures in corresponding colors.\u003c/p\u003e\n\u003cp\u003e(O) GST pull-down assays were performed using GST or the indicated IFI204 fragments fused with GST. The top panel shows schematics of the bait proteins used in the pull-down assays. The bottom panel shows the results of the GST pull-down assays. n = 3\u003c/p\u003e\n\u003cp\u003eData indicate means ± SD. *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8641308/v1/ad58577f0ba7c23407211156.png"},{"id":100954364,"identity":"3fca6a26-e780-49e1-8211-04d226ab208e","added_by":"auto","created_at":"2026-01-23 07:25:00","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":736831,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSTING is required for IFI204-induced neuronal pyroptosis in cerebral ischemic injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Experimental flow chart.\u003c/p\u003e\n\u003cp\u003e(B) Representative images and quantitative analysis of GSDMD positive neurons in cortex and striatum 1d after MCAO (Scale bar = 50 μm). n = 6.\u003c/p\u003e\n\u003cp\u003e(C) Confocal fluorescence images stained for Tom20 and GSDMD with quantification (Scale bar = 50 μm). n = 5.\u003c/p\u003e\n\u003cp\u003e(D) Transmission electron micrographs show the abnormal ultrastructure of the neurons in the cerebral peri-infarct area of MCAO mice. The yellow arrows showed pyroptosis pores on the plasma membrane. n = 5.\u003c/p\u003e\n\u003cp\u003e(E) Representative images and quantification of 8-OHdG staining neurons in cortex and striatum. n = 5.\u003c/p\u003e\n\u003cp\u003e(F, G) Immunoblots were probed for indicated proteins in cytosolic, mitochondrial and whole cell lysate. n = 5.\u003c/p\u003e\n\u003cp\u003e(H) Cytoplasmic TFAM, ACO2 and Cyto c levels as analyzed by Western blot. n = 5.\u003c/p\u003e\n\u003cp\u003e(I) Immunoblots of mitochondrial fraction of AAV-IFI204 infected MCAO mice brain under STING KO condition or not. n = 5.\u003c/p\u003e\n\u003cp\u003eData indicate means ± SD. ns, not significant. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-8641308/v1/d93ac123f41faf96a4489d3f.png"},{"id":100954362,"identity":"ead39220-810f-48b6-83d5-24a2bfb015f1","added_by":"auto","created_at":"2026-01-23 07:25:00","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2532205,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic of IFI204-driven neuronal pyroptosis via GSDMD-dependent mitochondrial permeabilization in ischemic stroke\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIschemic stroke induces mitochondrial oxidative damage, leading to release of mtDNA into the cytoplasm. Cytosolic mtDNA exclusively activates IFI204, which associates with STING via its pyrin domain to drive inflammasome activation. The activated inflammasome cleaves GSDMD, and GSDMD-N translocates to the mitochondrial membrane to form pores. These GSDMD-mediated pores further exacerbate mitochondrial damage characterized by reduced mitochondrial membrane potential (ΔΨm) and elevated ROS, and facilitate the release of additional mtDNA, Cyto c, and ACO2 into the cytoplasm, ultimately promoting neuronal pyroptosis.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-8641308/v1/35df53487f0ebfe399ec6232.png"},{"id":101298901,"identity":"afe3c484-680f-468a-8267-61d7f9945bf2","added_by":"auto","created_at":"2026-01-28 09:37:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16046646,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8641308/v1/c5933aa6-4ad9-4124-9944-a5fae1d2f33e.pdf"},{"id":100954361,"identity":"35a91e60-8c4a-4a77-940b-9471ecf45685","added_by":"auto","created_at":"2026-01-23 07:25:00","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":7219250,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-8641308/v1/161657d227461f349b56e609.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"IFI204 Drives Gasdermin D–Mediated Mitochondrial Permeabilization to Amplify Neuronal Pyroptosis in Ischemic Stroke","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIschemic stroke remains a leading cause of disability and mortality worldwide. Recanalization therapy constitutes the cornerstone of acute stroke management; however, despite expanded therapeutic time windows and advances in endovascular techniques, its effectiveness is limited by futile recanalization and reperfusion injury, resulting in suboptimal outcomes in a subset of patients\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Neuroinflammation is central to post-stroke pathology and represents a dynamically initiated and persistently sustained response to ischemic insult. This self-amplifying inflammatory cascade not only exacerbates secondary damage but also impairs acute-phase tissue repair, thereby representing a promising therapeutic target\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe innate immune system plays a pivotal role in orchestrating neuroinflammation and programmed cell death by initiating cerebral defense responses through the recognition of damage-associated molecular patterns (DAMPs)\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. This immune surveillance is not restricted to specialized immune cells; neurons also express functional pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and NOD-like receptors (NLRs), which detect these danger signals and trigger inflammatory storm\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Pyroptosis, a form of regulated lytic cell death, represents a canonical downstream outcome of PRRs activation. This process is executed through gasdermin D (GSDMD)-mediated perforation of the plasma membrane, leading to cell lysis and release of DAMPs and inflammatory cytokines\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Clinically, elevated plasma levels of pyroptosis-linked cytokines, including interleukin 1 beta (IL-1β) and interleukin 18 (IL-18), correlate with neurological deficit severity and predict poor outcomes in stroke patients\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. In animal models, GSDMD, the executor of pyroptosis, localizes to the plasma membrane of vulnerable neurons, indicating sites of cell rupture\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Emerging evidence demonstrates that the GSDMD N-terminal fragment (GSDMD-N) exhibits a stronger binding affinity for mitochondrial cardiolipin than for plasma membrane phospholipids\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, and localizes to mitochondria prior to translocation to the plasma membrane\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. The preferential mitochondrial localization of GSDMD-N explains why mitochondrial injury precedes plasma membrane rupture and establishing mitochondrial dysfunction as an early hallmark of pyroptosis. Subsequent GSDMD-mediated mitochondrial permeabilization results in the release of multiple DAMPs, including mitochondrial DNA (mtDNA), adenosine triphosphate (ATP), cytochrome c (Cyto c), and reactive oxygen species (ROS). Among these, mtDNA functions as a potent DAMP. Its hypomethylated CpG motifs, which resemble those found in bacterial DNA, enable mtDNA to activate innate immune pathways such as TLRs and inflammasomes\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. As a result, a self-amplifying loop is formed, linking cellular damage to sustained inflammatory signaling. Although the release of mtDNA following a stroke has been well-documented\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e, it remains unclear whether neuronal mtDNA release is directly mediated by GSDMD on mitochondrial membranes.\u003c/p\u003e \u003cp\u003eInterferon activated gene 204 (IFI204), along with its human homolog interferon gamma-inducible protein 16 (IFI16), are key members of the hematopoietic interferon-inducible nuclear protein with a 200-amino-acid repeat (HIN-200) family. IFI204 serves as an intracellular DNA sensor that binds double-stranded DNA and participates in transcriptional regulation and innate immunity responses\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Its C-terminal HIN domains can specifically recognize both pathogen-derived and self-DNA. Upon DNA binding, IFI204 undergoes conformational changes and oligomerization, allowing its N-terminal pyrin domain to recruit the ASC adaptor and promote inflammasome assembly, ultimately leading to caspase-1 activation and pyroptosis\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. In addition, IFI204-mediated DNA sensing functionally interacts with stimulator of interferon genes (STING) signaling, in which IFI204 acts as an upstream regulator of type I interferon production, thereby establishing a coordinated immune network\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Studies in myocardial infarction have demonstrated that IFI16/IFI204 promotes cardiomyocyte pyroptosis by sensing mtDNA, and that IFI204 knockdown alleviates cardiac damage in mice\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. However, the role of IFI204 in regulating mtDNA-driven inflammation and pyroptosis in ischemic stroke remain poorly defined.\u003c/p\u003e \u003cp\u003eTherefore, we propose that neuronal IFI204 serves as the central mediator that recognizes mtDNA and driving a vicious cycle involving pyroptosis, mitochondrial damage, and mtDNA release. This study aims to elucidate the functional interplay between the mtDNA\u0026ndash;IFI204 axis and STING signaling in ischemic stroke.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Transgenic mice\u003c/h2\u003e \u003cp\u003eNeuron-specific IFI204 knockout (IFI204\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e) mice were generated by crossing Ifi204 floxed mice with NestinCre transgenic mice. The Ifi204 floxed allele, containing loxP sites flanking exons 2 to 4, was generated using CRISPR/Cas9 technology by GemPharmatech LLC (Jiangsu, China). The Nestin\u003csup\u003eCre\u003c/sup\u003e mice, which express Cre recombinase under the control of the Nestin promoter on a C57BL/6J background, were obtained from Shanghai Model Organisms Center, Inc. Besides, wide-type (WT) C57BL/6J mice and and Sting1 knockout mice were purchased from GemPharmatech LLC (Jiangsu, China). All mice were housed under specific pathogen-free conditions in a controlled environment (20\u0026ndash;22\u0026deg;C, 12-h light/dark cycle) with food and water ad libitum. Male and female mice aged 6 to 8 weeks were used for subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Genotyping\u003c/h2\u003e \u003cp\u003eMouse genotyping was performed by polymerase chain reaction (PCR) analysis of genomic DNA. Tail biopsies were collected, and DNA was extracted using a standard alkaline lysis method\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. PCR amplification was conducted using a BIO-GENER GE4852T thermocycler (BIO‐GENER, China) with primer sets synthesized by Sangon Biotech. Primers specific for the \u003cem\u003eIfi204\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox\u003c/em\u003e\u003c/sup\u003e, and Nestin\u003csup\u003eCre\u003c/sup\u003e transgenes were listed in Supplementary Table\u0026nbsp;1. The resulting PCR products were separated on a 3% agarose gel and visualized under ultraviolet light after staining with SYBR Green.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 MCAO Model\u003c/h2\u003e \u003cp\u003eFocal cerebral ischemia was induced via transient occlusion of the right middle cerebral artery (MCA) for 60 min, followed by reperfusion, as previously described\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Mice were anesthetized with isoflurane (1\u0026thinsp;~\u0026thinsp;1.5%) delivered in medical air. After a midline cervical incision, the right common carotid artery was temporarily ligated. A silicon-coated monofilament (Beijing Cinontech, China) was introduced through the external carotid artery and advanced into the internal carotid artery until occlusion of the right MCA. Successful occlusion and subsequent reperfusion were confirmed in each animal using Laser Speckle Doppler Flowmetry (PeriCam PSI Z; Perimed, Sweden). Only animals exhibiting a reduction in regional cerebral blood flow (rCBF) greater than 70% during occlusion were included in the study. Throughout the procedure, body temperature was maintained at 37.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u0026deg;C using a heating pad. All surgical procedures were performed by an experimenter blinded to the group allocations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Measurement of Brain Infarct and Oedema Volume\u003c/h2\u003e \u003cp\u003eCerebral infarct volume was quantified using 2,3,5-triphenyltetrazolium chloride (TTC; Sigma, USA) staining. Briefly, brain tissues were rapidly harvested and coronally sectioned into 6 slices with a thickness of 1 mm. The slices were incubated in 2% TTC solution at 37\u0026deg;C for 10 min in the dark, fixed with 4% paraformaldehyde (PFA), and subsequently photographed using Epson Perfection V19 Scanjet (Seiko Epson, Japan). The infarct area on each slice was measured using Image J software (National Institutes of Health, USA). The infarct volume was calculated as a percentage using the following formula: [(contralateral hemisphere volume\u0026thinsp;\u0026minus;\u0026thinsp;non-infarcted ipsilateral volume) / contralateral hemisphere volume \u0026times; 2] \u0026times; 100%. The extent of oedema in the ipsilateral hemisphere was calculated separately as: [(ipsilateral hemisphere volume\u0026thinsp;\u0026minus;\u0026thinsp;contralateral hemisphere volume) / (contralateral hemisphere volume \u0026times; 2)] \u0026times; 100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Neurobehavioral Tests\u003c/h2\u003e \u003cp\u003eNeurobehavioral assessments were performed before surgery and periodically up to 30 days after middle cerebral artery occlusion (MCAO) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Sensorimotor function was evaluated using a battery of tests, including the adhesive removal test, the corner test, and the modified Neurological Severity Score (mNSS). Long-term learning and memory functions were assessed using the novel object recognition (NOR) test and the Morris water maze test, following established protocols. Testing was conducted in a controlled environment with consistent lighting and noise levels, and the experimenter was blinded to the group assignments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eModified Neurological Severity Score Test\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNeurological function was assessed using mNSS, a composite evaluation of motor, sensory, reflex, and balance functions. The mNSS scale ranges from 0 to 18, with 0 indicating normal neurological deficit and 18 indicating most severe neurological impairment. The assessment included tests of spontaneous activity, symmetry of limb movement, forepaw outstretching, climbing, body proprioception, and response to vibrissae touch\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRotarod Test\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMotor coordination and balance were evaluated using an automated rotarod apparatus. Prior to surgery, all mice underwent a two-day training and baseline testing protocol. During each session, the rotational speed increased linearly from 4 to 40 revolutions per minute over a 5-min period. Mice that fell within 5 s of the start were immediately placed back on the rod for another attempt. Each mouse performed three trials per day with a minimum inter-trial interval of 10 min. The longest latency to fall from the three trials was recorded as the baseline performance. Postoperativel testing was conducted at designated time points using the same acceleration protocol. The apparatus was cleaned with 70% ethanol between animals.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAdhesive Removal Test\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe Adhesive removal test was used to evaluate the sensitivity and dyspraxia of the forepaw\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Adhesive tapes measuring 3 \u0026times; 3 mm\u003csup\u003e2\u003c/sup\u003e were attached to the hairless area of the right forepaw. The time required for each mouse to detect and remove the tapes were recorded. If a mouse failed to touch or remove the tapes within 120 s, the time was recorded as 120 s. Mice were trained once daily for three consecutive days before surgery, and the performance on the final training day was recorded as the baseline value.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNovel Object Recognition Test\u003c/b\u003e The NOR test was conducted in an open-field arena between postoperative days 21 and 23. Mice were first habituated to the empty arena for 30 mins on two consecutive days. Twenty-four hours after the final habituation session, each mouse underwent a 5 min familiarization phase in which two identical objects were placed in symmetrical positions within the arena. After a 1 h interval, one familiar object was replaced with a novel object for a 5 min test phase. The arena and objects were cleaned with 75% ethanol between trials to eliminate olfactory cues. All sessions were video-recorded, and exploratory behavior, defined as directing the nose toward an object within a distance of approximately 2 cm, was analyzed using Smart v3.3.06 software (Panlab, Harvard Apparatus). The recognition index was calculated as the percentage of time spent exploring the novel object relative to the total exploration time for both objects.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMorris water maze test\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMice were subjected to Morris water maze testing beginning on day 24 after MCAO in a circular white pool with a diameter of 1.2 m and a height of 0.5 m, filled with water maintained at 22\u0026ndash;25\u0026deg;C \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. A hidden escape platform (10 cm diameter) was submerged 1 cm below the water surface in a predefined target quadrant. The test consisted of three phases. During the pre-training phase, the platform was made visible with an external cue, amd mice completed four 60 s trials. Mice that failed to locate the platform were gently guided to it. During the acquisition phase, the platform was hidden, and mice performed four 60 s trials per day for five consecutive days. Animals that did not locate the platform were guided to it and allowed to remain for 15 s to observe spatial cues. The daily mean escape latency and swimming path length was recorded. Finally, a probe trial was conducted on day 30 with the platform removed. Each mouse was allowed to swim freely for 60 s, and the time spent in the target quadrant, the number of crossings over the former platform location, and the overall swimming speed were recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Virus Production and Stereotaxic Injection\u003c/h2\u003e \u003cp\u003eRecombinant adeno-associated viruses (AAVs) targeting neurons were used, including a control virus (rAAV-hSyn-MCS-3FLAG-EGFP, AAV-NC) and a virus overexpressing IFI204 (rAAV-hSyn-IFI204-3FLAG-EGFP, AAV-IFI204), both with titers of at least 2.00 \u0026times; 10\u0026sup1;\u003csup\u003e3\u003c/sup\u003e v.g./mL (GeneChem, Shanghai, China). Viral sequences are listed in Supplementary Table\u0026nbsp;2. A total volume of 5 \u0026micro;L of virus was stereotaxically injected into right lateral ventricle at the following coordinates: coordinates: anterior-posterior, \u0026minus;\u0026thinsp;0.2 mm; medial-lateral, \u0026minus;\u0026thinsp;1.0 mm; dorsal-ventral, \u0026minus;\u0026thinsp;2.2 mm from bregma. Following a three -week period to allow sufficient gene expression, transient focal cerebral ischemia was induced by MCAO.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Transmission Electron Microscopy\u003c/h2\u003e \u003cp\u003eTissue specimens from the ipsilateral infarct border zone were sequentially fixed in 2.5% glutaraldehyde and 1% osmium tetroxide. After dehydration and embedding, ultrathin sections (50\u0026ndash;60 nm) were prepared and imaged using a transmission electron microscope (JEM-1400; JEOL, Japan). Mitochondrial morphology was analyzed by quantifying mitochondrial number and measuring their length. Mitochondrial counts were performed manually, length measurements were conducted using Image J software. Mitochondria displaying ultrastructural damage, characterized by loss of cristae and/or disruption of mitochondrial membrane, were identified as damaged.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Cell Culture and Treatment\u003c/h2\u003e \u003cp\u003eThe HT-22 immortalized mouse hippocampal neuronal cell line was obtained from Procell (Wuhan, China) and cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum at 37\u0026deg;C in a humidified incubator with 5% CO₂. Oxygen-glucose deprivation and reperfusion (OGD/R) was induced as previously described\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Briefly, cells were rinsed with phosphate-buffered saline (PBS) and incubated in glucose-free DMEM (Gibco, USA). Subsequently, the cells were transferred to a sealed anaerobic chamber (Billups-Rothenberg), which was equilibrated with a gas mixture of 95% N₂ and 5% CO₂ to induce OGD for 6 hours. Control cells were maintained under normoxic conditions (95% air, 5% CO₂). After the OGD period, cells were returned to the normoxic incubator, and the medium was replaced with standard glucose-containing DMEM to initiate reperfusion.\u003c/p\u003e \u003cp\u003eNeurons were treated with pharmacological agents immediately before and after OGD. Necrosulfonamide (NSA; MedChemExpress, USA) was dissolved in dimethyl sulfoxide (DMSO) and applied at a final concentration of 2 \u0026micro;M. Mitoquinone (MitoQ; MedChemExpress, USA) was dissolved in DMSO and applied at a final concentration of 1 \u0026micro;M. An equivalent volume of DMSO was administered to the vehicle control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Small RNA (siRNA) Interference\u003c/h2\u003e \u003cp\u003eHT-22 cells were cultured in antibiotic-free medium without penicillin and streptomycin until approximately 30% confluency was reached. IFI204 siRNA and the negative control siRNA were purchased from Sangon Biotech (Shanghai, China), and the corresponding sequences are listed in Supplementary Table\u0026nbsp;2. HT-22 cells were transfected with siRNA using GenMute\u0026trade; siRNA Transfection Reagent (SignaGen Laboratories, USA) according to the manufacturer\u0026rsquo;s instructions\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Cytosolic mtDNA Extraction and Quantification\u003c/h2\u003e \u003cp\u003eFor mtDNA analysis, cells were divided into two equal aliquots. One aliquot was lysed in 300 \u0026micro;L of 50 mM NaOH by boiling at 95\u0026deg;C for 30 min to solubilize total DNA, followed by neutralization with 30 \u0026micro;L of 1 M Tris-HCl (pH 8.0). This whole-cell lysate was used as a normalization control for total mtDNA quantification. The second aliquot was permeabilized in approximately 300 \u0026micro;L of buffer containing 150 mM NaCl, 50 mM HEPES (pH 7.4), and 25 \u0026micro;g/mL digitonin (EMD Chemicals, USA) with rotation at room temperature for 10 min. The homogenate was then centrifuged three times at 980 g for 3 min at 4\u0026deg;C to pellet intact cells. The resulting supernatant (cytoplasmic fraction) was further clarified by centrifugation at 17,000 g for 20 min to remove residual debris. Quantitative PCR (qPCR) was performed on both the whole-cell lysate and the cytoplasmic fraction. Primers targeting nuclear DNA (Tert) and mtDNA (D-loop, Cox1, Cox3 and ND1) were used, with their sequences detailed in Supplementary Table\u0026nbsp;1. The mtDNA abundance (Ct value) obtained from the whole-cell lysate was used to normalize the mtDNA levels measured in the cytoplasmic fraction\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 scRNA-seq data processing\u003c/h2\u003e \u003cp\u003eThe single-cell RNA sequencing dataset (GSE227651) was obtained from the Gene Expression Omnibus (GEO) database. Samples from the Sham and MCAO_D1 groups were selected for analysis. Raw sequencing reads were aligned to the mouse reference genome (mm10) and quantified using CellRanger software (version 7.1.0). Subsequent data processing and analysis were performed with the Seurat R package (version 4.3.0). Cells were subjected to standard quality control filtering, with exclusion criteria including fewer than 200 or more than 5,000 detected genes or a mitochondrial gene proportion exceeding 10%. Gene expression was normalized and variance-stabilized using the SCTransform method. To mitigate batch effects between samples, data integration was performed using the IntegrateData function. Dimensionality reduction was conducted using principal component analysis (PCA), followed by graph-based clustering. For visualization, t-distributed stochastic neighbor embedding (t-SNE) was applied using the top principal components via the RunTSNE function.\u003c/p\u003e \u003cp\u003eDifferential expression analysis between the Sham and MCAO_D1 groups was performed specifically within annotated neuronal clusters, using the FindMarkers function in Seurat. Identified differentially expressed genes (DEGs) were intersected with the MitoCarta3.0 database to obtain a neuron-enriched mitochondrial gene set. The expression pattern of these gene across samples was visualized in a heatmap. Gene Set Variation Analysis (GSVA) was applied to calculate single-cell enrichment scores for the gene set, and score distributions were compared between groups. Gene Set Enrichment Analysis (GSEA) was subsequently performed to evaluate coordinated regulation at the group level.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 RNA sequencing\u003c/h2\u003e \u003cp\u003ePublicly available human stroke transcriptomic data (GSE162955) were obtained from the GEO database. GSEA was performed on this dataset (infarct core vs. contralateral tissue) using the clusterProfiler R package to test for the enrichment of the cytosolic DNA-sensing pathway in the infarct core.\u003c/p\u003e \u003cp\u003eTotal RNA was extracted using TRIzol reagent (Thermo Fisher, USA) and subjected to quality control, with inclusion criteria of RNA concentration greater than 50 ng/\u0026micro;L, RNA integrity number greater than 7.0, and total RNA amount exceeding 1 \u0026micro;g. Strand-specific sequencing libraries were prepared from poly(A)-selected mRNA. Libraries were sequenced on an Illumina NovaSeq\u0026trade; 6000 platform using paired-end 150 bp reads. Raw sequencing data were processed with Cutadapt to remove adapters and low-quality bases. Clean reads were aligned to the mouse reference genome (Ensembl v112) using Hisat2. Transcript assembly and expression quantification (in FPKM) were performed using StringTie.\u003c/p\u003e \u003cp\u003eDifferential expression analysis was carried out using DESeq2 for datasets with biological replicates, with thresholds set at |log₂ (fold change)| \u0026ge; 1.2 and an adjusted p-value (q-value)\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Downstream analyses included functional enrichment analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, GSEA, and protein-protein interaction network construction using STRING. All analyses were performed using the OmicStudio cloud platform.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Molecular docking\u003c/h2\u003e \u003cp\u003eThe protein-protein interaction between IFI204 and STING was investigated through molecular docking. Amino acid sequences of mouse IFI204 and STING were retrieved from the UniProt database. The three-dimensional structure of the IFI204-STING complex was predicted using AlphaFold 3 in a fully automated mode. The top-ranked predicted complex model was subjected to energy minimization using the AMBER24 software package with the ff14SB force field. The binding affinity of the energy-minimized complex was then evaluated using the online PRODIGY tool. The interaction model exhibiting the most favorable binding energy was visualized and analyzed using PyMOL 2.5.3. In the resulting structural representation, IFI204 and STING are displayed as cartoon models in wheat and violet, respectively, with key interacting residues highlighted as sticks in the corresponding colors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 GST pull-down assay\u003c/h2\u003e \u003cp\u003eFor the glutathione-S-transferase (GST) pull-down assay, DNA fragments encoding full-length or truncated mouse IFI204, including amino acids residues 1\u0026ndash;88, 213\u0026ndash;413, and 417\u0026ndash;615, were cloned into the pET-GST vector to generate GST fusion constructs. These constructs were expressed in E. coli BL21. The resulting GST fusion proteins were affinity-purified using glutathione-Sepharose beads. For the binding assay, purified GST fusion proteins immobilized on beads were incubated with lysates derived from the cerebral ischemic penumbra for 4 h at 4\u0026deg;C with gentle agitation. After extensive washing, bound proteins were eluted and analyzed by Coomassie Brilliant Blue staining and immunoblotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Immunoblot\u003c/h2\u003e \u003cp\u003eFor total protein extraction, cells or brain tissues were lysed using RIPA buffer. Cytoplasmic and mitochondrial proteins were isolated using a commercial Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China) and Mitochondrial Protein Extraction Kit (Proteintech, China) respectively, according to the manufacturer's instructions. Protein samples were separated by SDS-PAGE on 8\u0026thinsp;~\u0026thinsp;15% gels and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk prepared in Tris-buffered saline (TBS) for 1 hour at room temperature and then incubated with primary antibodies overnight at 4\u0026deg;C. After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Immunoreactive bands were visualized using enhanced chemiluminescence reagent (Willget biotech, China) and detected with Azure 500 (Azure Biosystems, USA). Band intensities were quantified using ImageJ software. Details of the primary antibodies used are provided in Supplementary Table\u0026nbsp;3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16 Immunostaining\u003c/h2\u003e \u003cp\u003eImmunofluorescence staining was performed on both brain tissue sections and cultured HT-22 cells. For tissue preparation, mice were deeply anesthetized and transcardially perfused with PBS, followed by 4% PFA. Brains were harvested, post-fixed in 4% PFA overnight at 4\u0026deg;C, cryoprotected in 30% sucrose until sinking, and sectioned into 20 \u0026micro;m coronal slices using a cryostat (Leica, Germany). HT-22 cells were grown on confocal dish. All samples were blocked for 1 hour at room temperature in PBS containing 10% donkey serum, 1% bovine serum albumin (BSA), and 0.1% Triton X-100. Samples were then incubated with primary antibodies (Supplemental Table\u0026nbsp;3) overnight at 4\u0026deg;C. After three washes with PBS, samples were incubated with appropriate fluorophore-conjugated secondary antibodies (Supplemental Table\u0026nbsp;3) for 2 hours at room temperature, followed by nuclear counterstaining with DAPI for 15 min. Tissue sections were mounted with Fluoromount (Sigma, USA). Images were acquired using a confocal microscope (Olympus FV3000, Japan), and fluorescence signals were quantified using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17 FJC Staining\u003c/h2\u003e \u003cp\u003eFluoro-Jade C (FJC) staining was used to identify degenerating neurons\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. According to the instructions provided with the FJC ready-to-dilute staining kit (Biosensis, Australia), frozen brain sections mounted on gelatin-coated slides were dried at 50\u0026ndash;60\u0026deg;C for 40 min, and immersed in 70% ethanol for 5 min, followed by three rinses in distilled water. Sections were oxidized in a 0.06% potassium permanganate solution for 10 min and then rinsed in distilled water for 1 min to terminate oxidation. Subsequently, sections were stained with FJC working solution (0.0004% in 0.1% acetic acid) for 10 min. Slides were washed three times with distilled water, air-dried at room temperature, and coverslipped with a non-aqueous mounting medium for imaging.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.18 Detection and quantification of cytosolic DNA release\u003c/h2\u003e \u003cp\u003eMitochondria in live HT-22 cells were labeled with 200 nM MitoTracker Deep Red (Beyotime, China). at 37\u0026deg;C for 30 min. After fixation and immunofluorescent staining for DNA and DAPI, cytosolic DNA foci were quantified by manually counting extranuclear DNA puncta located outside both nucleus and the perimeter of MitoTracker-labeled mitochondria\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Cells containing more than two such extramitochondrial DNA foci were considered positive for cytosolic DNA release.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e2.19 Mitochondrial membrane potential\u003c/h2\u003e \u003cp\u003eMitochondrial membrane potential was evaluated using the JC-1 fluorescent probe (Mitochondrial Membrane Potential Assay Kit, Beyotime, China). According to the manufacturer's protocol, cells were incubated with JC-1 working solution. In polarized mitochondria, JC-1 forms aggregates emitting red fluorescence, whereas mitochondrial depolarization leads to monomeric forms emitting green fluorescence. Fluorescence images of both red and green signals were acquired using a confocal microscope and analyzed with Image J software. The ratio of red to green fluorescence intensity was calculated as an index of mitochondrial membrane potential.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e2.20 MitoSOX staining\u003c/h2\u003e \u003cp\u003eMitochondrial superoxide levels in HT-22 cells were assessed using the MitoBright ROS Deep Red fluorescent probe (Dojindon, Japan). Cells were cultured on confocal dishes, and a 10 \u0026micro;mol/L working solution was prepared by diluting the DMSO stock solution in culture medium. After removal of the growth medium, cells were incubated with the working solution at 37\u0026deg;C for 30 min, and subsequently washed. Fluorescence images were acquired using a confocal microscope equipped with a 561 nm laser and a 640\u0026ndash;700 nm emission filter.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e2.21 Statistics\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and analyzed using GraphPad Prism software (version 9.0, La Jolla, CA, USA). Normality of data distributions was verified via the Shapiro-Wilk test. For comparisons between two group, two-tailed unpaired Student\u0026rsquo;s t-test was applied for normally distributed data, whereas the Mann-Whitney U test was applied for non-normally distributed data. Survival analysis was performed using the log-rank (Mantel-Cox) test. Comparisons among multiple groups were conducted using one-way or two-way analysis of variance (ANOVA) followed by Š\u0026iacute;d\u0026aacute;k's multiple comparisons test for normally distributed data, or the Kruskal-Wallis test for non-normally distributed data. Repeated-measures data (e.g., behavioral tests) were analyzed using repeated-measures two-way ANOVA, followed by Tukey\u0026rsquo;s post hoc test for multiple comparisons as appropriate. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Neuronal IFI204 is upregulated after acute brain ischemia\u003c/h2\u003e \u003cp\u003eTo investigate the innate immune response following ischemic insult, we analyzed the publicly available human stroke transcriptomic dataset GSE162955. GSEA revealed that genes associated with cytosolic DNA-sensing pathway were significantly enriched in the infarct core (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). We next employed a mouse model of MCAO to examine alterations in this pathway, where differential expression analysis identified \u003cem\u003eIfi204\u003c/em\u003e as being significantly upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). IFI204 protein expression was evaluated by immunoblotting of peri-infarct tissue and by immunostaining of coronal brain sections from sham-operated mice and MCAO mice at 6 h, 1d, 3d, 5d, and 7d after MCAO (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u0026ndash;G). IFI204 expression exhibited dynamic changes over time following ischemic stroke. As Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD depicts, IFI204 expression increased after MCAO, reaching a peak of approximately 3.1-fold at 1 d compared with the control, followed by a gradual decline. Immunofluorescence staining confirmed the western blot results, showing a marked increase in IFI204 expression in neurons located in the peri-infarct region at 1 d after MCAO (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G). In addition, IFI204 signal displayed minor colocalization with GFAP (astrocytes marker), Iba1 (microglia and monocyte-derived macrophages marker), or CD31 (endothelial cells marker) (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA and B).\u003c/p\u003e \u003cp\u003eTo further validate these findings in vitro, HT-22 cells were subjected to OGD/R, and immunoblot analysis was performed at 6, 12, and 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). IFI204 protein was detectable under all conditions, with expression peaked 12 h after OGD/R and declining at 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Immunofluorescence analysis demonstrated that IFI204 was present in both the control and OGD/R-treated cells and was predominantly localized in the nucleus. However, following OGD/R, a portion of IFI204 signal was detected in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI), suggesting its potential role in cytoplasmic innate immune responses under ischemic conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Neuron\u0026ndash;specific IFI204 deletion attenuates ischemic stroke\u003c/h2\u003e \u003cp\u003eTo further determine the role of neuronal IFI204 in ischemic injury, neuron-specific IFI204\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e mice were generated by crossing IFI204\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice with Nestin\u003csup\u003eCre\u003c/sup\u003e mice (Fig. S2A and B). Immunofluorescence staining confirmed the selective deletion of IFI204 protein in neurons (NeuN\u003csup\u003e+\u003c/sup\u003e cells), with no observable change in non-neuronal colocalization (Fig. S2C). The decrease in IFI204 levels in the of IFI204\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e mice was confirmed by immunoblot analysis (Fig. S2D).\u003c/p\u003e \u003cp\u003eInfarct volume was examined by TTC staining 24 h after MCAO (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B). Successful induction of MCAO was verified by reduced blood flow during occlusion, as measured by laser Doppler flowmetry (Fig. S3A). To assess whether neuron-specific IFI204 deletion confers sex-dependent benefits, MCAO was also performed in female mice. Both male and female IFI204\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e mice exhibited reduced infarct size and oedema volume compared with littermate controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, and D; Fig. S3D). Neurodegeneration was evaluated by quantifying FJC\u003csup\u003e+\u003c/sup\u003e neurons in the cortex and striatum of the ipsilateral hemisphere 1 d after sham or MCAO surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Sham-operated IFI204\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e and IFI204\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e mice displayed similarly low levels of FJC\u003csup\u003e+\u003c/sup\u003e cells. After MCAO, a marked increase in FJC\u003csup\u003e+\u003c/sup\u003e cells was observed in both regions. Notably, in the cortex, IFI204\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice exhibited a significantly higher density of degenerating neurons than IFI204\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e mice following ischemia. In contrast, striatal neuronal degeneration did not differ significantly between the two groups post‑MCAO (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These data indicate that neuronal IFI204 deletion partially attenuates ischemia‑induced neuronal death.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Deletion of neuronal IFI204 improves neurological function\u003c/h2\u003e \u003cp\u003eTo evaluate neurological deficits after ischemic damage, a battery of behavioral tests was performed to comprehensively examine sensorimotor functions over a 30-day period after MCAO (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The mNSS, rotarod, and adhesive tape removal tests are applied to examine the sensorimotor functions of adult male mice. Mice subjected to MCAO showed pronounced sensorimotor impairments lasting at least 4 weeks. In contrast, neuron-specific IFI204 deletion significantly ameliorated sensorimotor dysfunction, as evidenced by lower mNSS scores, longer time to fall in the rotarod test, and shorter response time for adhesive removal from the contralateral forepaw (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u0026ndash;D). Cognitive function was assessed using the NOR test at 21 d post-stroke. Both MCAO groups demonstrated impaired performance; however, the preference index was higher in the IFI204\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e group than in the IFI204\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F). Spatial learning and memory were further assessed using the Morris Water Maze (MWM) test (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). During acquisition phase, IFI204\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice exhibited progressive reductions in escape latency and path length, whereas IFI204\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e mice displayed relatively shorter escape latency and swimming path length (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, I). During the probe trial, IFI204\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e mice spent more time in the target quadrant compared with IFI204\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice, although the number of platform crossings did not differ significantly between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ, K). Importantly, swimming speed showed no significant difference between the two MCAO groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL), indicating comparable visual and motor abilities in IFI204\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e and IFI204\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e mice during behavioral testing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Neuron-specific IFI204 knockdown reduces pyroptosis and pyroptotic mitochondrial damage\u003c/h2\u003e \u003cp\u003eGSDMD is the critical executor of pyroptosis. Neuronal pyroptosis in the cerebrum, including the cortex and striatum, was assessed by double immunofluorescence staining for GSDMD and NeuN. IFI204 knockout efficiently abrogated GSDMD expression after MCAO, as indicated by a marked reduction in the number of GSDMD\u003csup\u003e+\u003c/sup\u003e neurons in cortex, whereas no significant difference was observed in striatum (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Consistent with these findings, IFI204 deletion also diminished the GSDMD signal colocalized with mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Transmission electron microscopy revealed that MCAO induced plasma membrane disruptions and mitochondrial abnormalities, characterized by shortened, swollen mitochondria with disorganized cristae (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Compared with IFI204\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice after MCAO, IFI204\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e mice exhibited a partial attenuation of these structural injuries, as quantified by increased mean mitochondrial length and a reduced proportion of damaged mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The number of mitochondria per cell did not differ significantly between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Subsequently, mitochondrial and cytosolic fractions were analyzed by immunoblotting to assess the distribution of full-length GSDMD and GSDMD-N, as well as the release of mitochondrial components including cytochrome c (Cyto c; mitochondrial intermembrane space) and aconitase 2 (ACO2; mitochondrial matrix) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E). Concurrently, we examined the expression of mitochondrial transcription factor A (TFAM), a key regulator of mtDNA stability and mitochondrial function\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Disruption mitochondrial integrity can lead to aberrant TFAM expression. Western blot analysis demonstrated that MCAO significantly increased the levels of GSDMD-N in both cytosolic and mitochondrial fractions, accompanied by cytosolic accumulation of Cyto c, ACO2, and TFAM compared to sham-operated controls. These coordinated alterations were markedly attenuated in IFI204\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e mice subjected to MCAO, indicating that IFI204 deletion alleviated pyroptosis activation and mitochondrial membranes disruption after ischemic stroke (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E). scRNA-seq (GSE227651) data from animal models revealed a general downtrend among mitochondria-associated genes in neurons following MCAO, as determined by GSEA (Fig. S4A-D). This transcriptional response, involving genes related to mitochondrial quality control, the electron transport chain (ETC), and oxidative phosphorylation, likely reflects a mitochondrial stress response to acute ischemic injury. Given that altered mitochondrial membrane permeability compromises ETC electron transfer, we focused on two ETC components, ubiquinol-cytochrome c reductase core protein 1 (UQCRC1) and cytochrome c oxidase subunit 8A (COX8A). Western blot confirmed that COX8A was upregulated after MCAO, whereas this effect was suppressed by IFI204 knockout (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), suggesting that attenuation of pyroptosis‑driven membrane damage mitigates the acute mitochondrial stress response. Consistent with the alleviated mitochondrial damage, oxidative DNA damage was reduced, as evidenced by a significant decrease in 8‑OHdG\u003csup\u003e+\u003c/sup\u003e neurons in IFI204‑deficient mice post‑MCAO (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Loss of IFI204 in HT-22 triggers mtDNA release during pyroptosis via GSDMD pore formation\u003c/h2\u003e \u003cp\u003eIn order to further validate the role of IFI204 during neuronal pyroptosis in vitro, IFI204 expression was silenced using siRNA (Fig. S5A). Knockdown efficiency was confirmed by qRT-PCR and immunoblotting (Fig. S5B, C). Immunoblot analysis of subcellular fractions showed that OGD/R increased the GSDMD-N levels in both cytosolic and mitochondrial fractions, an effect attenuated by IFI204 knockdown, suggesting that IFI204 promotes both GSDMD activation and its mitochondrial translocation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). This observation was further supported by a reduction in the co-localization of GSDMD-N with MitoTracker following IFI204 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). As mitochondrial GSDMD-N pores can dissipate membrane potential and trigger the release of mtDNA and mitochondrial reactive oxygen species (mtROS)\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, we next assessed these downstream events. Cytosolic double-stranded DNA (dsDNA) was defined by the absence of co-localization with DAPI and MitoTracker signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). OGD/R increased the number of cytoplasmic dsDNA foci per cell, accompanied by elevated mitochondrial superoxide levels, as detected by the mitoSOX probe, and a shift from red (J-aggregates) to green (monomers) fluorescence in JC-1 staining, indicating mitochondrial membrane depolarization. These alterations were partially reversed by IFI204 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-I). We hypothesized that the cytoplasmic dsDNA originated from leaked mtDNA. Co-immunostaining for mitochondrial markers TFAM, and dsDNA following OGD/R revealed mitochondrial origin of the cytosolic DNA foci (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). Furthermore, qPCR analysis of isolated cytosolic DNA demonstrated an increased mtDNA copy number after OGD/R, which was reduced by IFI204 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK). Collectively, these findings indicate that IFI204 regulates mtDNA leakage, likely by facilitating GSDMD pore formation on mitochondrial membranes. This conclusion is further supported by Western blot results showing that IFI204 knockdown preserved mitochondrial membrane integrity, as evidenced by reduced cytosolic leakage of mitochondrial components, including TFAM, Cyto c, and ACO2, and by suppression of respiratory chain components COX8A upregulation after OGD/R (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL, M).\u003c/p\u003e \u003cp\u003eWe next hypothesized that mtDNA released through GSDMD pores could amplify pyroptosis by activating the cytosolic DNA‑sensing pathway involving IFI204. Indeed, exogenous mtDNA stimulation after OGD/R further increased IFI204 expression, supporting the existence of a feedforward loop (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eN). To delineate the role of GSDMD pores in mediating mtDNA release, we applied NSA, a specific inhibitor of GSDMD oligomerization. NSA treatment effectively suppressed pyroptosis, as indicated by reduced cleavage of GSDMD and caspase‑1 (Fig. S6A). Mitochondrial damage induced by OGD/R was partially attenuated, as reflected by restored TFAM expression and decreased GSDMD‑N levels in mitochondrial fractions (Fig. S6A-B). These effects were accompanied by a reduction in cytosolic mtDNA release after OGD/R (Fig. S6C-E). Furthermore, western blot and immunofluorescence analyses demonstrated that NSA treatment downregulated the expression of the cytosolic DNA sensor IFI204 (Fig. S6F, G). Together, these results support a self-amplifying loop in which mtDNA released via GSDMD pores activates IFI204, thereby further amplifing the pyroptotic cascade.\u003c/p\u003e \u003cp\u003eTo investigate whether attenuation of mitochondrial oxidative stress could prevent mtDNA leakage, we employed MitoQ, a mitochondrial-targeted antioxidant reported to protect mtDNA from oxidative damage and stabilizing TFAM\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. In HT22 cells subjected to OGD/R, MitoQ treatment significantly reduced mitochondrial superoxide levels, as measured by mitoSOX staining, while exerting no effect on basal levels in control cells (Fig. S7A, C). Concurrently, MitoQ markedly decreased both the proportion of cells containing cytoplasmic dsDNA foci and the number of dsDNA foci per cell following OGD/R (Fig. S7B, D, E). These findings demonstrate that MitoQ protects against OGD/R-induced mtDNA leakage in neurons, likely by scavenging mtROS and preserving mtDNA integrity. In addition, MitoQ treatment suppressed the OGD/R-induced upregulation of IFI204 protein (Fig. S7F).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Re-expression of neuronal IFI204 aggravates neuronal pyroptosis and mitochondrial damage\u003c/h2\u003e \u003cp\u003eWe next sought to determine whether supplementation of neuronal IFI204 could exacerbate mitochondrial damage and neuronal pyroptosis post-stroke. IFI204 was re-expressed in C57BL/6J IFI204\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e and IFI204\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e mice via stereotactic injection of an adeno-associated virus (AAV), with negative control (AAV-NC) treated mice serving as controls, three weeks prior to MCAO induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Under the hSyn promoter, the viruses with EGFP signals were predominantly observed in NeuN\u003csup\u003e+\u003c/sup\u003e neurons, and IFI204 expression was successfully restored by AAV9-hSyn-Ifi204 delivery (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Notably, neuronal IFI204 re-expression in MCAO mice promoted neuronal pyroptosis and partially reversed the protective phenotype. Specifically, the number of GSDMD\u003csup\u003e+\u003c/sup\u003e neurons in the peri-infarct cortex at 24 h after MCAO was substantially increased by elevated neuronal IFI204 levels in both IFI204\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e and IFI204\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e MCAO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), whereas no difference was detected in the striatum (Fig. S8A). Immunoblot and immunofluorescence co‑localization analyses collectively demonstrated that AAV-mediated IFI204 reconstitution increased GSDMD-N levels in both cytosolic and mitochondrial fractions and enhanced its co-localization with mitochondria markers (Tom20) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, H, I). Furthermore, IFI204 reconstitution also abolished the reduction in oxidative DNA damage in IFI204\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e mice, with a concomitant elevation in the count of 8‑OHdG⁺ neurons, while no significant change was observed in IFI204\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, Fig. S8B). As expected, ultrastructural analysis by electron microscopy revealed that transfection with AAV9-hSyn-Ifi204 aggravated abnormal mitochondrial morphology, leading to a reduction in mitochondrial length per neuron, and an increased proportion of damaged mitochondria characterized by swelling and disorganized cristae (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, G). Stroke increased cytoplasmic expression of TFAM, Cyto c and ACO2, which was further exacerbated by IFI204 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). Meanwhile, the mitochondrial level of COX8A was amplified (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). Collectively, the specific aggravation of injury-associated hallmarks upon IFI204 re-expression provides direct genetic evidence that IFI204 is a central driver of GSDMD-mediated mitochondrial dysfunction and neuronal pyroptosis after ischemic stroke.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e3.7 IFI204 binding to STING via pyrin domain medicates neuronal pyroptosis and mitochondrial damage\u003c/h2\u003e \u003cp\u003eTranscriptomic analysis using RNA sequencing was performed to elucidate the underlying mechanisms and potential molecular targets through which IFI204 regulates neuronal pyroptosis under ischemic condition. A total of 1,150 DEGs were identified, including 227 upregulated and 923 downregulated genes, compared with IFI204\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e MCAO group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The key mediators \u003cem\u003eGsdmd\u003c/em\u003e and \u003cem\u003eSting1\u003c/em\u003e were among the downregulated genes. GO enrichment analysis of biological processes revealed that the DEGs were predominantly enriched in immune-related terms, with \"immune system process,\" \"innate immune response,\" and \"inflammatory response\" ranking among the most significantly enriched terms (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). GSEA further supported these observations by revealing significant positive enrichment of the \"cytoplasmic pattern recognition receptor signaling pathway\" and \"pyroptosis\" gene sets in control samples relative to IFI204\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e MCAO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Consistently, unsupervised heatmap analysis of DEGs within these gene sets showed a consistent downregulation pattern, prominently involving \u003cem\u003eGsdmd\u003c/em\u003e and \u003cem\u003eSting1\u003c/em\u003e, in the IFI204\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). KEGG pathway analysis of the top enriched pathways highlighted the \"NOD-like receptor signaling pathway\" (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Subsequent GSEA confirmed this result and additionally demonstrated significant positive enrichment of the \u0026ldquo;cytosolic DNA-sensing pathway\u0026rdquo; in the IFI204\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e group relative to IFI204\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). Heatmap visualization of these two key innate immune pathways revealed that \u003cem\u003eIfi204\u003c/em\u003e, \u003cem\u003eGsdmd\u003c/em\u003e, and \u003cem\u003eSting1\u003c/em\u003e clustered as core DEGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). To further delineate functional interactome within the enriched pathway, a protein\u0026ndash;protein interaction (PPI) network was constructed using DEGs annotated to the KEGG \u0026ldquo;NOD‑like receptor signaling pathway.\u0026rdquo; A high-confidence interaction between Ifi204 and Sting1 was identified, with a STRING confidence score greater than 900 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). Finally, Western blot analysis of STING protein expression in the same samples confirmed its significant downregulation in the IFI204\u003csup\u003e\u003cem\u003ecKO\u003c/em\u003e\u003c/sup\u003e group, thus validating the transcriptional changes at the protein level and linking them to the observed pyroptotic phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003eTo validate the in vivo findings and further explore the cell-intrinsic mechanisms, we performed RNA sequencing on HT-22 neuronal cells subjected to OGD following transfection with either si-NC or si-IFI204. Differential expression analysis confirmed marked transcriptional reprogramming upon IFI204 knockdown (Fig. S9A). Analysis of the top 25 mitochondrial-associated GO terms revealed a pronounced enrichment of processes related to mitochondrial membrane integrity and dysfunction (Fig. S9B). Specifically, terms including \u0026ldquo;mitochondrial inner membrane,\u0026rdquo; \u0026ldquo;mitochondrial outer membrane,\u0026rdquo; \u0026ldquo;regulation of mitochondrial membrane potential,\u0026rdquo; \u0026ldquo;negative regulation of mitochondrial membrane permeability,\u0026rdquo; and \u0026ldquo;mitochondrial outer membrane permeabilization\u0026rdquo; were highly enriched. Critically, enrichment of term \u0026ldquo;positive regulation of cytochrome c release from mitochondria\u0026rdquo; provided direct transcriptional support for our biochemical observation that IFI204 deficiency reduces cytosolic Cyto c accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), linking the transcriptomic changes to the suppression of mitochondrial permeabilization. KEGG pathway analysis identified the \u0026ldquo;NOD-like receptor signaling pathway\u0026rdquo; as one of the most significantly enriched pathways (Fig. S9C). GSEA further confirmed significant positive enrichment of both the \u0026ldquo;NOD-like receptor signaling pathway\u0026rdquo; and the \u0026ldquo;cytosolic DNA-sensing pathway\u0026rdquo; in control cells, consistent with observations in brain tissue (Fig. S9D). \u003cem\u003eSting1\u003c/em\u003e is an important and core transcriptional co-activator in the NOD-like receptor signaling pathway (Fig. S9E). Finally, a PPI network constructed from genes within the NOD-like receptor signaling pathway again predicted a direct interaction between Ifi204 and Sting1, reinforcing this key molecular across both in vivo and in vitro models (Fig. S9F).\u003c/p\u003e \u003cp\u003eThe interaction between IFI204 and STING suggested by transcriptomic analyses was further examined at the protein level. In brain tissue from MCAO mice, immunofluorescence staining revealed co-localization of IFI204 and STING within NeuN\u003csup\u003e+\u003c/sup\u003e neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ, K). Similarly, in OGD-treated HT-22 cells, IFI204 co-localized with STING in MAP2\u003csup\u003e+\u003c/sup\u003e neuronal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eL, M). To define the structural domains responsible for IFI204\u0026ndash;STING interaction, molecular docking analysis was performed, revealing a potential interaction between IFI204 and STING, with a predicted binding energy of -9.9 kcal/mol. The model indicated that residues within the N-terminal segment of IFI204, corresponding to its Pyrin domain (amino acids 1\u0026ndash;88), formed hydrogen bonds with STING (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eN). To test this prediction biochemically, a GST pull-down assay was conducted. Recombinant, purified GST-tagged full-length IFI204 successfully pulled down endogenous STING from post-MCAO brain lysates. This interaction was specifically mediated by the Pyrin domain of IFI204, as a GST-tagged Pyrin domain construct also bound STING, whereas GST-tagged HIN-A or HIN-B domains did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eO).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Genetic ablation of STING partially rescues neuronal damage exacerbated by IFI204 overexpression\u003c/h2\u003e \u003cp\u003eGiven the upstream regulation of IFI204 on STING in neurons, AAV-IFI204 was stereotactically delivered into the lateral ventricle of WT and Sting1-knockout (STING KO) mice, followed by MCAO induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Successful ablation of STING was confirmed by Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG). Immunofluorescence analysis of the cortex and striatum revealed a STING-dependent effect of IFI204 on neuronal pyroptosis. In the cortex, IFI204 overexpression significantly increased the number of GSDMD\u003csup\u003e+\u003c/sup\u003e neurons in WT mice, whereas this effect was partially attenuated in STING KO mice. In contrast, no significant differences were observed in GSDMD\u003csup\u003e+\u003c/sup\u003e neuron counts in the striatum among the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Immunofluorescence analysis further showed that the co‑localization of GSDMD‑N with the mitochondrial marker Tom20 was greatly enhanced in WT mice overexpressing IFI204, whereas this enhancement was substantially attenuated in STING KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Besides, ultrastructural analysis by transmission electron microscopy indicated that cortical neurons from IFI204-overexpressing WT mice exhibited membrane disruption, reduced mitochondrial number and average length, and an increased proportion of damaged mitochondria. These pathological alterations were ameliorated in STING KO neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). Similarly, IFI204 overexpression led to a marked increase in the number of neurons exhibiting oxidative DNA damage, defined as 8-OHdG and NeuN double-positive cells in WT mice, which was partially mitigated in STING KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). Reduced protein expression of molecules associated with pyroptosis and mitochondrial damage was also detected in STING KO mice receiving AAV-IFI204 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF-I). These findings indicate that STING is required for IFI204-induced pyroptotic mitochondrial damage.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn the current study, we demonstrated that IFI204 deficiency attenuates neuroinflammation following MCAO. Genetic ablation of IFI204 reduced neuronal pyroptosis, alleviated mitochondrial damage and mtDNA leakage, and ultimately improved neurological outcomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Mechanistically, we identified a direct interaction between IFI204 and STING, mediated by the PYD of IFI204, which facilitates the membrane translocation and pore-forming activity of GSDMD, a key executioner of pyroptosis. The GSDMD pores subsequently formed on mitochondrial membranes serve as the direct conduit for mtDNA release into the cytosol. The leaked mtDNA further amplifies this cascade by activating the IFI204-STING axis, thereby establishing a self-sustaining inflammatory loop. Deletion of IFI204 disrupts this loop by suppressing STING-driven signaling, ultimately protecting ischemic neurons.\u003c/p\u003e \u003cp\u003eThis study identifies the p200 family protein IFI204 as a crucial DNA sensor linking hypoxic or ischemic injury to neuroinflammation. We demonstrate that IFI204 expression was upregulated in neurons following ischemic stroke, likely triggered by fragmented DNA released from damaged tissue. Another important finding is the subcellular redistribution of IFI204, which is predominantly nuclear under physiological conditions but partially translocated to the cytoplasm post-OGD. This redistribution appears to be functionally relevant, reflecting a shift from a nuclear DNA surveillance role to a cytoplasmic DNA-sensing function\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Regulation of this nucleo-cytoplasmic shuttling involves specific post-translational modifications, including nuclear localization signal (NLS) deacetylation-mediated nuclear import and chromosome region maintenance 1 (CRM1)-dependent nuclear export\u003csup\u003e[\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Importantly, subcellular localization influences downstream signaling outcomes. Nuclear IFI204 or IFI16 is associated with interferon-beta production, while cytoplasmic accumulation preferentially promotes inflammasome assembly\u003csup\u003e[\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Our observation of cytoplasmic IFI204 linked it to pyroptosis is consistent with its established role in activating the ASC/Caspase-1 inflammasome.\u003c/p\u003e \u003cp\u003eThe functional versatility of IFI204 is reflected in its mechanistic diversity in DNA recognition. IFI204 directly senses dsDNA in a non-sequence-specific manner via electrostatic interactions mediated by its C-terminal HIN domains\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. This direct sensing is further enhanced by the cooperative assembly of DNA-bound complexes into higher-order filaments, driven by the N-terminal PYD domain\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. In addition to direct recognition, IFI204 also engages in indirect DNA sensing within the nucleus as part of the DNA Damage Response (DDR) pathway, detecting genotoxic stress through protein\u0026ndash;protein interactions with factors such as breast cancer type 1 susceptibility protein (BRCA1) and histone H2B \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBuilding on its DNA-sensing capability, IFI204 or IFI16 orchestrates pyroptosis through a complex network of synergistic pathways. IFI204 functions as a core inflammasomes component and directly nucleates its assembly. Inflammasome formation is further amplified through cooperative interactions with other innate immune molecules. For example, in a myocardial infarction model, IFI204 or IFI16 enhances inflammasome assembly efficiency by interacting with galectin-3, thereby amplifying the pyroptotic response\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Similarly, IFI204 acts through STING to drive pyroptosis during traumatic brain injury\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e, consistent with our observations in neurons. Beyond inflammasome assembly, IFI204 has been shown to interact specifically with TLR4 via the PYD region, facilitating its dimerization and enhancing nuclear factor kappa B (NF-κB) signaling, which leads to increased expression of essential pyroptosis components such as pro-IL-1β\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. Moreover, IFI204 induces mitochondrial disorders, promoting ROS accumulation and exacerbating pyroptotic responses\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Mitochondrial dysfunction also intensifies inflammation by releasing mtDNA, which is sensed with high affinity by the IFI16 rs6940 variant, thereby activating the IFI16\u0026ndash;caspase-1 inflammasome\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNotably, IFI204 or IFI16 directly interfaces with the cGAS-STING signaling pathway. IFI16 has been shown to recruit cGAS and enhance cGAMP synthesis, thereby facilitating STING activation\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e, or to function as a cGAS cofactor through its dsDNA-binding capacity\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. Alternatively, STING recruitment by IFI16 or IFI204 activates downstream TBK1, leading to phosphorylation of IRF3 and NF-κB\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. Depletion of IFI16 under dsDNA or 2'3'-cGAMP stimulation impairs STING phosphorylation\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Our RNA sequencing analysis revealed that IFI204 modulates STING signaling in neurons. Genetic knockout of STING suppressed its expression and partially attenuated IFI204 overexpression-induced neuronal pyroptosis. GST pull-down assays confirmed direct binding between IFI204 and STING, localizing the interaction to the PYD of IFI204. Consistent with our findings, previous co-immunoprecipitation studies in HSV-1-infected or DNA-stimulated human monocytes have demonstrated the PYD of IFI16 mediates its interaction with STING\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. Mutations within this domain disrupted IFI16-STING complex formation in 293T cells\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e, suggesting that further identification of critical binding residues within the PYD of IFI204 is warranted. However, such studies are limited in their ability to exclude indirect interaction. Recent work in a traumatic brain injury model showed that SUMO-specific protease 7 (SENP7) directly interacts with IFI204 and catalyzes its deSUMOylation\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. This modification enhances STING activation and strengthens IFI204\u0026ndash;STING binding. In our earlier study, STING-mediated microglial pyroptosis involved the NLRP3 inflammasome pathway\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Therefore, the IFI204-STING axis in neurons may not function in isolation, suggesting potential cross-talk between IFI204-inflammasome and NLRP3 inflammasome that warrants further investigation.\u003c/p\u003e \u003cp\u003eOur data identify GSDMD as a key molecular bridge linking pyroptotic signaling to mitochondrial impairment in neurons. Recent research have shown that gasdermin proteins target mitochondrial membranes through specific lipid interactions, with the N-terminal domain of GSDMD exhibiting high-affinity binding to cardiolipin and oligomerizing into 10\u0026ndash;18 nm pores\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. Disruption of both inner and outer mitochondrial membranes results in mitochondrial dysfunction, increased ROS production, and the release of mtDNA and cytotoxic proteins, thereby establishing mitochondrial damage as a critical irreversible step in pyroptosis execution\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. This mechanism was validated in our neuronal model, in which treatment with the GSDMD oligomerization inhibitor NSA significantly reduced cytosolic mtDNA release following OGD, confirming GSDMD pore formation as a central event in neuronal mitochondrial damage during pyroptosis. Similarly, GSDME-NT, generated by caspase-3 cleavage, also targets mitochondria via cardiolipin binding, promoting mtDNA and cytochrome c release\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. mtDNA leakage thus serves as a critical pathological link between mitochondrial damage and sterile inflammation, a process that is further amplified by downstream DNA sensors such as IFI204 and STING.\u003c/p\u003e \u003cp\u003eThe release process requires mitochondrial outer membrane permeabilization, which can be initiated through several distinct mechanisms under stress conditions. Some mechanisms depend on the formation of functional membrane pore structures. For example, voltage-dependent anion channel 1 (VDAC1) oligomerization disrupts mitochondrial membrane integrity and promotes mtDNA release\u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Alternatively, pathological opening of the mitochondrial permeability transition pore (MPTP) can cause collapse of mitochondrial membrane potential and increase outer membrane permeability, thereby providing a physical channel for mtDNA escape\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. During apoptosis, mitochondrial outer membrane permeabilization occurs through macropore formation in a BAX/BAK dependent manner, allowing herniation of the inner mitochondrial membrane into the cytoplasm and subsequent mtDNA release\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. In parallel, studies have shown that during necroptosis, phosphorylated mixed lineage kinase domain-like protein (MLKL) translocates to and permeabilizes mitochondrial membranes, promoting mtDNA release in a microtubule-dependent manner\u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. Other mechanisms do not rely on direct pore formation but instead involve mitochondrial structural remodeling, abnormal mtDNA packaging or vesicle-mediated transport. For instance, abnormal mitochondrial fission induced by dynamin-related protein 1 (DRP1), accompanied by excessive mitochondrial ROS explosion, compromises DNA integrity and membrane permeability, indirectly facilitating mtDNA release\u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e. Furthermore, deficiency in TFAM disrupts normal mtDNA packaging and stability, resulting in abnormal mtDNA copy number and cytoplasmic accumulation. Notably, the mitochondrial E3 ligase mitochondrial-anchored protein ligase (MAPL) initiates a unique pathway by packaging mtDNA into mitochondrial-derived vesicles\u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. These vesicles are transported to lysosomes where GSDME pore formation triggers lysosomal rupture, ultimately releasing mtDNA through a mechanism that bypasses direct mitochondrial membrane perforation.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eOverall, this study delineates a novel inflammatory cascade involved in ischemic neuronal injury. Hypoxic stress induces upregulation of IFI204, which directly interacts with STING, promoting inflammasome assembly, and leading to caspase-1 activation and GSDMD cleavage. The resulting GSDMD-NT forms pores on mitochondria membranes, facilitating mtDNA release. This mtDNA release further amplifies activation of the IFI204-STING-GSDMD axis, creating a feed-forward loop of inflammation and pyroptosis. These findings identify neuronal IFI204 as a central node integrating DNA sensing, innate immune signaling, and mitochondrial damage, and suggest potential therapeutic targets for mitigating neuroinflammation in ischemic stroke.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eAAV\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAdeno-associated virus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eACO2\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAconitase 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eBRCA1\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBreast cancer type 1 susceptibility protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eBSA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBovine serum albumin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCOX8A\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCytochrome c oxidase subunit 8A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCyto c\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCytochrome c\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDAMP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDamage-associated molecular pattern\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDEG\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDifferentially expressed gene\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDMEM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco\u0026rsquo;s Modified Eagle Medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDMSO\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDimethyl sulfoxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDRP1\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDynamin-related protein 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003edsDNA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDouble-stranded DNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eETC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eElectron transport chain\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eFJC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFluoro-Jade C\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGO\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Ontology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGSEA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Set Enrichment Analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGST\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlutathione-S-transferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGSDMD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGasdermin D\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGSDMD-N\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGasdermin D N-terminal fragment\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGSVA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Set Variation Analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHIN\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHematopoietic interferon-inducible nuclear protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHRP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHorseradish peroxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eIFI16\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterferon gamma-inducible protein 16\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eIFI204\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterferon activated gene 204\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eIL-1β\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterleukin 1 beta\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eIL-18\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterleukin 18\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eKEGG\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKyoto Encyclopedia of Genes and Genomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMAPL\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrial-anchored protein ligase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMCA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMiddle cerebral artery\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMCAO\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMiddle cerebral artery occlusion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMLKL\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMixed lineage kinase domain-like protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003emNSS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eModified Neurological Severity Score\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMWM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMorris water maze\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMitoQ\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitoquinone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMPTP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrial permeability transition pore\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003emtDNA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrial DNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003emtROS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrial reactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eNF-κB\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNuclear factor kappa B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eNLR\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNOD-like receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eNSA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNecrosulfonamide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eNOR\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNovel object recognition\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eOGD/R\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOxygen-glucose deprivation and reperfusion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePBS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePCA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePrincipal component analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePFA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eParaformaldehyde\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePPI\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProtein-protein interaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePRR\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePattern recognition receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003erCBF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRegional cerebral blood flow\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eROS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003escRNA-seq\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSingle-cell RNA sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandard deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003esiRNA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSmall interfering RNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSTING\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStimulator of interferon genes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTLR\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eToll-like receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTFAM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrial transcription factor A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTTC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2,3,5-Triphenyltetrazolium chloride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eUQCRC1\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUbiquinol-cytochrome c reductase core protein 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eVDAC1\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVoltage-dependent anion channel 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eWT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWild-type\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments involving animals were conducted according to the ethical policies and procedures approved by the ethics committee of the First Affiliated Hospital of University of Science and Technology of China (2025-N (A)-279).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Nos. 82471310, 82471311, 82101368), the Anhui Provincial Natural Science Foundation (No. 2408085MH214).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePFX and NS: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing-Original Draft, Visualization. TQ and MYZ: Investigation, Methodology. RL, CRT and YYZ: Investigation. WH and PFX: Investigation, Resources, Writing-Review \u0026amp; Editing, Supervision, Project administration, Funding acquisition. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCHENG Z, WANG H R, GENG X K, et al. Time and Tissue Windows in Futile Reperfusion after Ischemic Stroke[J]. Aging Dis. 2025;16(5):2544\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXIE L, HE M, YING C D, et al. Mechanisms of inflammation after ischemic stroke in brain-peripheral crosstalk[J]. Front Mol Neurosci. 2024;17:1400808.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIADECOLAR C, BUCKWALTER M S ANRATHERJ. 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Nat Cell Biol. 2025;27(10):1708\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuroinflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jneu","sideBox":"Learn more about [Journal of Neuroinflammation](http://jneuroinflammation.biomedcentral.com)","snPcode":"12974","submissionUrl":"https://submission.nature.com/new-submission/12974/3","title":"Journal of Neuroinflammation","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"IFI204, Pyroptosis, GSDMD, Mitochondrial DNA (mtDNA), Ischemic stroke, Neuroinflammation","lastPublishedDoi":"10.21203/rs.3.rs-8641308/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8641308/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGasdermin D (GSDMD)\u0026ndash;mediated pore formation on mitochondrial membranes is known to exacerbate pyroptosis. The cytosolic DNA sensor interferon activated gene 204 (IFI204) can activate the inflammasome to induce pyroptosis. However, whether and how IFI204 regulates mitochondrial membrane permeabilization to drive pathological outcomes in ischemic stroke remains unclear. Here, using a mouse model of middle cerebral artery occlusion (MCAO), we demonstrate that IFI204 was predominantly expressed in neurons and increased to peak at 24 hours after ischemic injury. Neuron-specific deletion of IFI204 alleviated cerebral infarction, reduced neuronal degeneration, and restored long-term sensorimotor coordination and cognitive function. These protective effects correlated with attenuated neuronal pyroptosis and mitochondrial dysfunction, as evidenced by decreased levels of GSDMD N-terminal fragment (GSDMD-N) and reduced mitochondrial colocalization. Conversely, adeno-associated virus-mediated re-expression of IFI204 in knockout mice restores these pathological features. In vitro, IFI204 is both necessary and sufficient to trigger this cascade. Transcriptomic profiling revealed a significant downregulation of the stimulator of interferon genes (STING) within the NOD-like receptor signaling pathway in IFI204-deficient neurons. Mechanistically, glutathione S-transferase (GST) pull-down assays confirmed a direct interaction between the pyrin domain (PYD) of IFI204 and STING. This interaction triggers caspase-1 activation and GSDMD cleavage, generating GSDMD-N, which subsequently forms pores specifically on mitochondrial membranes. These pyroptotic pores disrupted mitochondrial integrity, exacerbating dysfunction, and facilitating the cytosolic release of mitochondrial DNA (mtDNA), cytochrome c, and aconitase 2. Notably, the released mtDNA further activated IFI204, establishing a pathogenic feed-forward cycle that exacerbates mitochondrial damage and inflammatory neuronal death. Genetic ablation of STING partially abrogated the pyroptosis-promoting effect of IFI204. Collectively, these findings demonstrate that IFI204-driven cytosolic mtDNA sensing underlies a neuronal inflammatory mechanism responsible for pyroptosis and mitochondrial damage in ischemic stroke.\u003c/p\u003e","manuscriptTitle":"IFI204 Drives Gasdermin D–Mediated Mitochondrial Permeabilization to Amplify Neuronal Pyroptosis in Ischemic Stroke","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-23 07:17:15","doi":"10.21203/rs.3.rs-8641308/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-23T22:26:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-23T22:22:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-15T06:55:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"94971330718796882123204631428896002966","date":"2026-02-01T21:13:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"37498974025716938913534898998524527354","date":"2026-01-22T02:49:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-21T14:44:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-21T13:29:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-21T00:52:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Neuroinflammation","date":"2026-01-19T15:28:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuroinflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jneu","sideBox":"Learn more about [Journal of Neuroinflammation](http://jneuroinflammation.biomedcentral.com)","snPcode":"12974","submissionUrl":"https://submission.nature.com/new-submission/12974/3","title":"Journal of Neuroinflammation","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1ba8a1a8-0707-4f94-abac-cd0cad5c7fec","owner":[],"postedDate":"January 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T12:55:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-23 07:17:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8641308","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8641308","identity":"rs-8641308","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}