CCCTC-binding factor (CTCF) is a highly conserved DNA-binding protein crucial for 3D genome organization and gene regulation. Previous studies have shown that CTCF overexpression in hepatocellular carcinoma (HCC) is associated with poor prognosis. This study aimed to elucidate the molecular mechanisms underlying CTCF's role in HCC pathogenesis and identify the downstream effectors that mediate its oncogenic functions. We generated CTCF knockout HCC cell lines (Huh7 and PLC5) using the CRISPR-Cas9 technology. CTCF knockout significantly reduced HCC cell proliferation, colony formation, migration, and invasion capabilities while inducing cellular senescence. Chromatin immunoprecipitation-sequencing analysis revealed that CTCF knockout in HCC cells preferentially affected its binding to promoters and enhancers rather than topologically associating domain boundaries. In contrast, genes that were commonly downregulated in the two CTCF knockout cell lines were significantly enriched in the energy metabolism pathway, and fatty acid desaturase 1 (FADS1) emerging as a key CTCF target gene. We further found that CTCF and FADS1 expression levels are highly correlated in clinical HCCs with high expression levels associated with poor patient survival. FADS1 knockdown recapitulated the CTCF knockout phenotypes, including reduced ATP levels, impaired glycolysis and oxidative phosphorylation, and decreased NAD+/NADH ratios. Re-expression of FADS1 in CTCF knockout cells significantly upregulated ATP level associated with enhanced glycolytic and respiratory capacity. In vivo xenograft studies confirmed that tumor growth was significantly inhibited by CTCF or FADS1 depletion. This study highlighted a CTCF-FADS1 axis that predominately regulates energy metabolism in HCC cells with profound prognostic and therapeutic implications for HCCs.
Hexokinase 2 (HK2), a rate-limiting glycolytic enzyme, regulates inflammatory signaling and inflammasome activation in multiple neurological diseases; however, its function in epilepsy remains largely unexplored. Here, we show that HK2 expression is markedly upregulated in the brains of pilocarpine-induced epileptic mouse model. Pharmacological inhibition or microglia-specific HK2 deletion inhibited microglia activation and enhanced microglial phagocytosis, and subsequently decreased both the frequency and cumulative duration of hippocampal paroxysmal discharges during the chronic phase of spontaneous epilepsy. HK2 deficiency significantly increased the expression of the microglial activation marker CD68, and elevated levels of the phagocytic receptor TREM2. TREM2 inhibitor TREM2-IN-1 (OPA) inhibited HK2 deletion-induced microglial phagocytosis and chronic recurrent seizures. Overall, these findings indicate that microglial HK2-dependent glycolytic reprogramming plays a key role in epilepsy progression and highlight HK2 as a potential therapeutic target.
Type I interferon (IFN) signaling is associated with detrimental outcomes during Mycobacterium tuberculosis (Mtb) infection, in part through antagonism of IL-1β-mediated protective immunity. Viperin, an IFN-stimulated gene induced downstream of type I IFN, has previously been implicated in suppressing innate immune responses during Mtb infection. However, whether Viperin modulates IL-1β-mediated protective pathways that contribute to host control of Mtb remains unclear. Viperin expression was robustly induced following Mtb infection in a type I IFN-dependent manner in bone marrow-derived macrophages and murine lungs. Genetic deletion of Viperin significantly reduced bacterial burden both in vitro and in vivo. Mechanistically, Viperin-deficient (Viperin−/−) macrophages exhibited enhanced IL-1β responses accompanied by increased COX-2 expression and elevated PGE2 production. These changes were associated with improved control of intracellular Mtb. The enhanced antibacterial activity of Viperin−/− macrophages was dependent on COX-2 activity, as treatment with the selective COX-2 inhibitor celecoxib significantly reduced bacterial restriction. Conversely, Viperin overexpression suppressed inflammatory mediator production and impaired macrophage control of Mtb. These findings identify Viperin as a functional downstream effector of type I IFN signaling that restrains IL-1β–associated antibacterial responses during Mtb infection. By linking Viperin to regulation of the IL-1β/COX-2/PGE2 pathway, our study provides mechanistic insight into how type I IFN signaling can undermine host defense in tuberculosis and supports further exploration of Viperin as a potential target for host-directed therapeutic strategies.
Glycine is a major inhibitory neurotransmitter in the central nervous system and plays a critical role in synaptic transmission and plasticity. Although its postsynaptic actions mediated by glycine receptors (GlyRs) are well characterized, the presynaptic mechanisms by which glycine regulates synaptic vesicle recycling, particularly endocytosis, remain poorly understood. In this study, we combined pharmacological manipulations with membrane capacitance measurements, calcium imaging, and pHluorin-based fluorescence assays to investigate this process. We found that 1 mM glycine inhibited both slow and rapid endocytosis at large calyx-type synapses and slowed vesicle retrieval at small, conventional hippocampal synapses. This inhibition was GlyR dependent and was associated with membrane depolarization and increased basal cytosolic Ca2+; pharmacological experiments further implicated Ca2+-sensitive downstream signaling. Pharmacological manipulation of protein kinase C (PKC) and phospholipase D (PLD) signaling further suggests that PKC/PLD-related signaling contributes to glycine-induced inhibition of vesicle retrieval. Our findings support a previously unrecognized role for glycine in regulating vesicle recycling and provide a framework for examining how GlyR-dependent ionic changes interact with Ca2+-sensitive membrane-trafficking processes.
Neurodegenerative diseases (NDDs) involve self-amplifying interactions among proteotoxic stress, mitochondrial dysfunction, and neuroinflammation. Emerging evidence increasingly implicates mitochondria-endoplasmic reticulum contact sites (MERCs) as dynamic homeostatic hubs that may link these processes, but their causal status in human NDDs remains incompletely established. This review synthesizes evidence connecting MERCs biology with NDD-associated mitochondrial stress, Ca2+ dysregulation, lipid remodeling, redox signaling, and inflammatory cell death. We frame the MERCs-centered model as a working, testable hypothesis and use Eleutherococcus senticosus as a case study for candidate phytochemical modulation of MERCs-associated stress pathways. Available cellular, animal, and human-tissue evidence suggests that altered MERCs structure and function can contribute to mitochondrial Ca2+ overload, reactive oxygen species (ROS) generation, mtDNA/DAMP release, and NLRP3 inflammasome activation. Natural products may influence this network indirectly through mitochondrial metabolism, membrane composition, ER stress, redox buffering, and regulated Ca2+ handling, but direct proof of MERCs restoration remains limited. MERCs should be viewed as a convergent homeostatic hub and therapeutic hypothesis rather than a validated upstream cause of NDDs. Future studies should distinguish structural contact-site changes from functional correction and should test phytochemical candidates using direct MERCs proximity, flux, lipidomics, and bioenergetic assays.
Post-stroke memory dysfunction is common in elderly people. We showed previously that a bone fracture (BF) shortly before ischemic stroke also caused long-lasting memory dysfunction in young mice, and inhibition of inflammation by α7-nicotinic acetylcholine receptor (α7-nAChR) agonist treatment alleviated their cognitive deficit. Excessive synapse removal by glial cells contributes to neuronal network disruption and neurobehavioral deficits during the acute and subacute stages of stroke. However, its role at the chronic stage of stroke remains unclear. In this study, we analyzed the impact of microglia- and astrocyte-mediated synapse removal at the chronic stage of ischemic stroke on memory function and synapse structure in young (2-month-old) and old mice (15-18-month-old). We also tested whether α7-nAChR agonist treatment reduced synapse loss in young mice with BF+stroke. Ischemic stroke was induced by permanent occlusion of the distal middle cerebral artery (dMCAO). The BF model was induced by creating a tibia fracture under aseptic conditions 6 h before dMCAO. An α7-nAChR agonist was administered immediately before BF and 1 day after dMCAO. Memory function was evaluated weekly for 8 weeks using the Y-maze test and the novel object recognition test one week before and 8 weeks after dMCAO. Atrophic volumes, synapse-engulfing astrocytes and microglia/macrophages, RNA expression profiles, neurite length, and dendritic spine density were analyzed in the peri-atrophic regions and hippocampal regions eight weeks after dMCAO. Old mice developed long-lasting memory dysfunction (> eight weeks), had larger atrophic volumes, shorter neurite length, and fewer dendritic spines, accompanied by increased synapse-engulfing astrocytes and microglia/macrophages in the peri-atrophic and hippocampal regions than young stroke-only mice. Old stroke mice showed higher upregulation of inflammatory pathways and more downregulation of neurite growth and synaptic transmission pathways than in young stroke-only mice. Young mice with BF+stroke had more synapse-engulfing astrocytes and microglia/macrophages in the hippocampal regions than in young stroke-only mice. An α7-nAChR agonist treatment reduced synapse-engulfing microglia/macrophages and astrocytes in the hippocampi of BF+stroke mice. Enhanced synapse elimination contributes to the long-lasting post-stroke memory dysfunction. Inhibition of neuroinflammation at the early stage of stroke can reduce synapse loss and improve post-stroke memory function.
Scar tissue impede axonal regeneration and hinder functional recovery after spinal cord injury. Previous studies have demonstrated excision of scar tissue during the subacute phase (particularly on day 10) not only enhances neuronal regeneration but also identifies microtubule stabilization as a key contributor to axonal regeneration in this process. Here we grafted fibrin scaffolds loaded with paclitaxel (a microtubule-stabilizing agent) immediately after scar excision during subacute stage, with the aim of stabilizing axonal microtubules. Our results showed that paclitaxel promoted functional recovery of injured rats by enhancing neuronal regeneration and axonal elongation post resecting scars in subacute stage. In paclitaxel-fibrin-grafted group, the microenvironment post injury was significantly improved. The secretion of pro-regenerative factors (laminin, BDNF and NT-3) was upregulated and the deposition of CSPG, a key inhibitory component of the scar tissue, was reduced in injured spinal tissue under paclitaxel-fibrin. Moreover, BSCB (blood spinal cord barrier) was reconstructed that alleviated the continuous infiltration of immune cells into the lesion site. Collectively, these findings suggest that the administration of paclitaxel following scar resection during subacute stage may hold significant potential in the treatment of SCI.
Anthracycline chemotherapy induces cardiotoxicity through mitochondrial damage and cell apoptosis that are exacerbated in vivo by angiotensin II (Ang II). Growth differentiation factor 15 (GDF15) is a stress-responsive cytokine with protective functions but its capacity to mitigate doxorubicin (Dox)-induced cardiotoxicity and Ang II-associated cardiorenal stress remains unclear. Human embryonic stem cell-derived cardiomyocytes were used to evaluate Dox toxicity and to benchmark recombinant GDF15 against conventional dexrazoxane pretreatment. Cardiomyocyte viability, mitochondrial integrity and function, apoptosis, and transcriptome profiles by RNA sequencing were evaluated. To determine in vivo efficacy, C57BL/6 J mice were administered GDF15 (0.5 mg/kg, intraperitoneal) 24 h before once weekly Dox (5 mg/kg). Ang II was infused subcutaneously via surgically implanted osmotic pump. Mice were sacrificed for histopathologic analysis 1 week after the fourth cycle of Dox. In vitro, Dox-induced mitochondrial fragmentation, loss of membrane potential, transcriptional dysregulation, and apoptosis were significantly attenuated by recombinant GDF15 pretreatment, with efficacy comparable to dexrazoxane. Cytotoxic effects of daunorubicin on cardiomyocytes were also attenuated by GDF15. In vivo, Ang II, Dox, and Dox + Ang II induced cardiorenal injury characterized by hypertension, sarcomere disarray, myocardial and renal fibrosis, and glomerular, tubular, and perivascular abnormalities. GDF15 prophylaxis mitigated these pathologic changes, preserved myocardial and renal tissue architecture, and reduced cardiorenal fibrosis. Recombinant GDF15 pretreatment can protect the heart and kidneys from Dox-induced cardiorenal toxicity and Ang II-exacerbated injury. These findings encourage further investigation into GDF15 as a potential prophylaxis against cardiorenal toxicity associated with anthracycline chemotherapy and renin-angiotensin system activation.
Gamma-glutamyl transferase (GGT) is a clinically used biomarker of hepatobiliary injury and alcohol exposure, yet accumulating evidence links higher GGT to systemic cardiometabolic and other comorbidities. Although large-scale GWAS have defined many GGT-associated loci in European-ancestry cohorts, the genetic architecture of GGT, its cross-trait liability, and the translational utility of GGT polygenic risk remain insufficiently characterized in East Asian populations. We performed a cross-biobank genetic analysis of GGT in East Asian populations. We conducted a GWAS of circulating GGT in 46,704 Han Taiwanese participants from the CMUH Biobank and integrated GWAS summary statistics from East Asian biobanks to quantify cross-trait genetic correlations with cardiometabolic phenotypes. Putative causal effects were evaluated using univariable and BMI-adjusted multivariable Mendelian randomization (MR) analyses. Translational utility was assessed by constructing a GGT polygenic risk score (PRS) and testing its associations across the clinical phenome and in BMI-stratified time-to-event analyses of incident type 2 diabetes (T2D). In a Han Taiwanese GWAS (n = 46,704), we identified 18 genome-wide significant loci for circulating GGT, including five putative previously unreported Han Taiwanese signals at RBMS3, APBB2, AC091905.2, CCDC88C, and PML after comparison with prior GGT GWAS and the GWAS Catalog. Across East Asian biobanks, GGT shared genetic liability with ALT/AST and cardiometabolic traits, including T2D, adiposity, blood pressure, and lipids. MR supported causal effects of higher GGT liability on ALT/AST, and T2D-related signals persisted after BMI-adjusted multivariable MR. A GGT PRS showed pleiotropic PheWAS associations and stratified incident T2D within BMI strata consistently. This cross-biobank study refined and extended the East Asian genetic architecture of circulating GGT and demonstrated broad shared liability with cardiometabolic traits, alongside BMI-independent causal signals for hepatic injury biomarkers and persisting T2D-related effects. These findings support the use of GGT genetics and polygenic risk for comorbidity stratification and genomics-informed risk prediction in precision medicine.
Cardiac arrest-induced brain injury significantly affects patient prognosis, yet effective therapeutic strategies remain lacking in clinical practice. In recent years, mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have garnered considerable attention for their notable neuroprotective potential. However, studies in cardiac arrest models remain relatively limited. This study aimed to investigate the protective effects and underlying mechanisms of human embryonic stem cell-derived mesenchymal stem cell extracellular vesicles (hESC-MSC-EVs) against brain injury following cardiac arrest/resuscitation (CA/CPR). In vivo, a rat model of cardiac arrest was established by asphyxia, and hESC-MSC-EVs were administered intravenously after resuscitation. In vitro, HT22 cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). The results demonstrated that hESC-MSC-EVs significantly improved neurological function at 24 h post-resuscitation, reduced serum levels of NSE and S100B, alleviated pathological damage, and inhibited apoptosis in the hippocampal CA1 region at 7 days after resuscitation. In vitro experiments further confirmed that hESC-MSC-EVs increased HT22 cell viability, reduced reactive oxygen species levels, and suppressed apoptosis. Further mechanistic investigation revealed that hESC-MSC-EVs significantly attenuated pyroptosis in HT22 cells and rat hippocampal neurons, and suppressed the expression of cGAS/STING pathway-related proteins. Activation of the cGAS/STING pathway exacerbated OGD/R-induced HT22 cell injury and CA/CPR-induced brain injury, whereas hESC-MSC-EVs intervention effectively reversed these effects. Mechanistically, miR-181a-5p was identified to target and inhibit Cgas and transfection with miR-181a-5p mimics downregulated the cGAS/STING signaling pathway and alleviated pyroptosis in HT22 cells. Collectively, hESC-MSC-EVs ameliorate brain injury following CA/CPR by inhibiting cGAS/STING-mediated neuronal pyroptosis, offering novel potential therapeutic targets and strategies for clinical management.
Diabetes mellitus erectile dysfunction (DMED) is frequently accompanied by corpus cavernosum (CC) fibrosis and endothelial dysfunction; however, the cellular drivers and molecular mechanisms underlying these pathological changes remain incompletely understood. Endothelial-to-mesenchymal transition (EndMT), a phenotypic conversion in which endothelial cells lose endothelial characteristics and acquire mesenchymal features, has emerged as a key contributor to tissue fibrosis under inflammatory and metabolic stress conditions. Macrophages (Mφ), the predominant immune cell population in CC tissue in diabetes, are major sources of pro-inflammatory cytokines and may promote fibrosis by driving EndMT. This study investigated the role and mechanisms of Mφ in CC fibrosis during DMED and explored potential therapeutic targets. In a streptozotocin-induced DMED rat model, erectile function was significantly impaired, as evidenced by a marked reduction in the maximum intracavernous pressure-to-mean arterial pressure (ICP-to-MAP) ratio. This functional deterioration resulted from pronounced Mφ infiltration and enhanced fibrotic remodeling in CC. Pharmacological depletion of Mφ using clodronate liposomes significantly restored erectile function and increased the smooth muscle-to-collagen ratio. In vitro, co-culture of corpus cavernosum endothelial cells (CCEC) with Mφ under high-glucose (HG) conditions impaired endothelial tube formation and sprouting capacity, accelerated wound closure, and induced EndMT, characterized by downregulation of endothelial markers (CD34 and VE-cadherin) and upregulation of mesenchymal markers (Vimentin and α-SMA). RNA sequencing identified enrichment of interleukin-1 (IL-1)–related responses and Nuclear factor kappa B (NF-κB) signaling during EndMT. Further analyses demonstrated that HG stimulated Mφ to upregulate and secrete interleukin-1 beta (IL-1β), which in turn induced EndMT in CCEC accompanied by activation of NF-κB signaling, as evidenced by increased phosphorylation of p65 and IκBα. Pharmacological inhibition of the IL-1 receptor or NF-κB signaling effectively attenuated EndMT in vitro. Notably, in vivo administration of the IL-1 receptor antagonist Anakinra significantly improved erectile function and suppressed EndMT-associated molecular changes in DMED rats. Mφ-derived IL-1β promotes cavernosal fibrosis and erectile dysfunction in diabetes by inducing EndMT through activation of the NF-κB pathway. Targeting the IL-1β/NF-κB/EndMT axis represents a promising therapeutic strategy for the treatment of DMED.
Human kidney development remains incompletely characterized at the level of chromatin accessibility. Here, we investigate chromatin accessibility dynamics (CAD) during the differentiation of human embryonic stem cells (hESCs) into kidney organoids (E-iKOs). Time-course ATAC-seq reveals progressive remodeling of pluripotency-associated chromatin landscapes toward an intermediate mesoderm state, followed by metanephric mesenchyme specification and early nephron epithelialization. These transitions are accompanied by increased accessibility at genomic regions enriched for binding motifs of transcription factors, including SIX, HOX, LHX, PAX, EBF, RFX, WT1, HNF1B, and TEAD. Functional analyses demonstrate that HNF1β is required for tubular differentiation, as HNF1β deficiency results in severe defects in tubule formation while podocytes remain detectable. Integrative analysis of chromatin accessibility and time-course transcriptomics further identifies the histone H3K79 methyltransferase DOT1L as a regulator required for nephron progenitor induction and epithelial differentiation. DOT1L deficiency is associated with widespread alterations in chromatin accessibility and gene expression, particularly at regions enriched for SIX, HOX, PAX, EBF, WT1, HNF1B, and TEAD motifs, accompanied by impaired mesenchymal-to-epithelial transition. Together, this study provides a valuable resource describing chromatin accessibility dynamics during human kidney organoid differentiation and a foundation for future studies of human nephrogenesis in vitro.
Recurrent implantation failure (RIF) remains a significant challenge in assisted reproductive technology, yet its underlying pathogenesis is poorly understood. Successful embryo implantation requires synchronization between the blastocyst and a receptive uterus. Mechanosensitive ion channel PIEZO1 has emerged as a key regulator in various physiological and pathological contexts. However, the role of PIEZO1 in embryo implantation remains unclear. This study aims to elucidate the role and molecular mechanism of PIEZO1 in embryo implantation and to explore its clinical relevance in patients with RIF. The expression levels of PIEZO1 in endometrial tissues from patients with RIF and healthy controls were analyzed by immunohistochemistry. A uterine-specific Piezo1 knockout mouse model was established to assess the role of PIEZO1 during embryo implantation. Ca2+ imaging was employed to determine PIEZO1 mediated Ca2+ influx in endometrial cells. Mechanical stretch was applied to study PIEZO1 regulated signaling pathway. mRNA sequencing of mice endometrium was applied to examine differential regulated pathways. Chromatin immunoprecipitation sequencing (ChIP-seq), ChIP-qPCR and co-immunoprecipitation (Co-IP) were employed to identify NFATc2 target genes and transcriptional partners downstream of PIEZO1. The effect of Nfatc2 silencing on embryo implantation was evaluated. PIEZO1 expression was significantly up-regulated in the human endometrium during the secretory phase compared to the proliferative phase. However, its expression was markedly reduced in patients with RIF in the secretory phase. Uterine conditional deletion of Piezo1 in mice resulted in subfertility due to impaired endometrial receptivity and implantation failure. Mechanistically, PIEZO1 deficiency reduced both Yoda1 and mechanical stretch-induced Ca2+ influx and NFATc2 activation. Consistent with this, in vivo knockdown or blockade of NFATc2 activation inhibited embryo implantation in mice. Further study revealed that NFATc2 regulates a subset of PGR target genes by forming a transcriptional complex with PGR. These findings highlight the critical role of PIEZO1 in uterine receptivity during embryo implantation through the Ca2+/NFATc2 axis. Defects in this pathway represent a potential mechanism underlying implantation failure, suggesting PIEZO1 as a promising therapeutic target for implantation failure.
The CRISPR/Cas7-11 system is a recently characterized RNA targeting tool that exhibits low toxicity, minimal off-target effects, and reduced collateral RNA cleavage. While Cas7-11 has been recently tested in zebrafish embryos as ribonucleoprotein (RNP) complexes, its use as mRNA–single guide RNA (sgRNA) formulations and its application in human embryonic stem cells (hESCs) have not been explored. In this study, we evaluated the efficiency and specificity of Cas7-11 in zebrafish embryos and hESCs. In zebrafish,injection ofCas7-11and sgRNA resulted in evident reductions of target transcripts, accompanied by clear phenotypic outcomes. ExogenousEGFPmRNA levels were reduced to 31.90
Danon disease (DD) is a rare X-linked lysosomal storage disorder that predominantly affects cardiac and skeletal muscles, characterized by glycogen and autophagic material accumulation. DD is caused by mutations in LAMP2, encoding a key mediator of lysosome-dependent autophagic degradation. A de novo nonsense mutation c.669 T > G (p. Tyr223Ter, LAMP2Y223*) was identified in a young DD patient. A knock-in mouse carrying the homologous mutation (Lamp2Y228*) successfully recapitulated the typical clinical features of DD, including a short PR interval and pre-excitation pattern. Transmission electron microscopy and immunofluorescence assays showed aberrant autophagolysosomes and glycogen accumulation in both cardiac and skeletal muscle tissues. Mechanistic studies in vivo and in vitro demonstrated the impaired chaperone-mediated autophagy (CMA) and increased glucose uptake through the CMA-dependent RAB10-GLUT4 pathway following mutant LAMP2 expression. These findings identify a RAB10-GLUT4 axis linking LAMP2 deficiency to the enhanced glucose uptake and further implicate this pathway as a contributor of glycogen accumulation in DD.
As global warming escalates, heat stress in both animals and humans has emerged as a pressing concern. As a physiological response to uncomfortable high ambient temperature, heat stress also significantly affects the emotional states of animals. The paraventricular thalamus (PVT), a pivotal brain region for stress response and emotion regulation, expresses many CRF receptors. However, the role of CRF receptors in the PVT in emotion regulation under heat conditions remains unclear. In our study, we revealed distinct sex-specific responses to heat conditions in mice: male mice presented significantly increased anxiety-like behavior, whereas female mice presented no significant alterations. After heat exposure, the male mice presented anxiety-like behavior, accompanied by a trend toward upregulation CRFR2 expression in the PVT and inhibition of the AKT/CREB signaling pathway. Conversely, reducing CRFR2 expression or inhibiting CRFR2 in the PVT effectively alleviated heat-induced anxiety-like behavior in the OFT. Furthermore, our findings revealed that chemogenetic activation of Vglut2 neurons in the PVT induced anxiety-like behavior primarily reflected in the OFT, whereas chemogenetic inhibition of these neurons mitigated heat-triggered anxiety in the OFT. Notably, the reduction in CRFR2 expression or the specific inhibition of CRFR2 in PVTVglut2 neurons diminished heat-induced anxiety-like behavior in the OFT. Thus, our findings demonstrate that the CRFR2 and activity of Vglut2 neurons within the PVT contribute to the development of anxiety-like behavior in the OFT following heat exposure in male mice.
Cytotoxic T lymphocyte (CTL)-mediated platelet destruction represents an important pathogenic mechanism in primary immune thrombocytopenia (ITP) patients. Rho-associated coiled-coil kinase 2 (ROCK2) is an emerging regulator of immune balance, but its role in pathogenic CTL activation in ITP remains undefined. Here, we demonstrated that selective ROCK2 inhibition with KD025 potently suppressed CTL-mediated platelet destruction. In vitro, KD025 treatment of CTLs from ITP patients suppressed key effector functions, reducing degranulation as measured by CD107a expression, diminishing the secretion of cytotoxic molecules such as granzyme B and perforin, and decreasing CTL-platelet conjugate formation, resulting in reduced platelet apoptosis and activation. RNA-sequencing revealed downregulation of cytotoxic and glycolytic programs, with enrichment of JAK-STAT signaling. Mechanistically, KD025 reversed the pathogenic metabolic shift in ITP CTLs by lowering glycolytic flux and restoring mitochondrial respiration, accompanied by decreased STAT3 phosphorylation. IL-6-mediated STAT3 activation largely reversed these effects, indicating a ROCK2-STAT3-dependent mechanism. In vivo, both daily KD025 administration to an active ITP mouse model and transplantation of KD025-pretreated CD8+ T cells into irradiated Rag1−/− mice alleviated CTL-mediated platelet apoptosis and increased platelet counts. Collectively, these findings supported a model in which ROCK2 contributed to STAT3-associated transcriptional and metabolic regulation of CTL pathogenicity, providing a rationale for further preclinical and translational evaluation of ROCK2 inhibition in ITP.
Vasoactive Intestinal Peptide (VIP) is a pleiotropic neuropeptide regulating diverse cellular and physiological processes. Its functions are primarily mediated through two G protein–coupled receptors, VPAC1 and VPAC2. The aim of this study was to perform an integrative analysis of VPAC receptor signalling, encompassing receptor–G protein coupling, second messenger production, kinase activation and transcriptional responses in T cells. Receptor interactions with Gα subunits were analysed using BRET assays. Stable Jurkat T-cell lines overexpressing VPAC1 (J-OEV1) or VPAC2 (J-OEV2) were generated. VPAC-dependent intracellular signalling in these cells was assessed by measuring cAMP and Ca2+ levels, performing phospho-kinase arrays and Western blot analyses, and evaluating immune mediator expression, cell viability, and proliferation. VPAC1 showed interaction with both Gαs and Gαq subunits, whereas VPAC2 preferentially interacted with Gαs. In Jurkat cells, both receptors overexpression enhanced cAMP signalling, while increased Ca2+ responses were restricted to VPAC1. In both J-OEV1 and J-OEV2 cells, VIP treatment reduced phosphorylation of inflammatory kinase-associated proteins. Overexpression of either receptor induced distinct basal transcriptional profiles of transcription factors and cytokines, which were further modulated by CD3/CD28-activation and VIP. While proliferation was not altered with overexpression, J-OEV2 showed a reduced redox metabolism at 72 h. This study aimed to identify functional differences between VPAC1 and VPAC2 signalling and reveals reproducible subtype-specific differences at early signalling. In Jurkat cells, both receptors induce a shift in its basal state; however, changes do not persist during TCR-driven activation, resulting in largely convergent effector responses.
Astrocytes play a significant role in neuroprotection by internalizing neurodegenerative aggregates and facilitating their degradation. Recent studies indicate that α-Synuclein (α-SYN) protofibrils promote the transfer of pathogenic aggregates and dysfunctional mitochondria between astroglia via tunneling nanotubes (TNTs), which enhances cell survival and resistance to apoptosis. However, the underlying mechanism of TNT-driven apoptosis resistance remains unclear. We find that α-SYN protofibrils induce aberrant mitochondria with decreased membrane potential (Ψm) and promote dynamic actin remodeling by relocating phosphorylated focal adhesion kinase (pFAK) to the nucleus, which triggers TNT formation in human astrocytoma cell lines and primary murine astrocytes. The important novel finding of this study is that pFAK in the nucleus co-localizes with Nanog, a crucial transcription factor for preserving stemness, and the interaction between pFAK and Nanog is critical for promoting p53 degradation via Mdm2-mediated ubiquitination and upregulating autophagy, thereby supporting the survival of astroglia exposed to toxic α-SYN protofibrils. ROCK inhibitor y-27632 also drives TNT-formation via pFAK translocation to the nucleus, colocalizes with Nanog, and enhances stemness-related gene expression. Inhibiting TNT with the actin depolymerizing agent cytochalasin-D prevents pFAK co-localization with Nanog in the nucleus and fails to protect cells from α-SYN-induced apoptosis. Nanog knockdown does not degrade p53 and hinders cell rescue from apoptosis. Furthermore, these transient TNTs transfer mitochondria to adjacent cells, potentially helping maintain metabolic stability. This study reveals that the TNT formation pathway promotes pFAK-Nanog interaction in the nucleus, leading to p53 degradation, which protects astroglia against α-SYN proteotoxicity and prevents apoptosis.
The roof plate that covers the dorsal neural tube is a key organizer or signaling center that generates morphogenetic signaling proteins to regulate neural crest cell production and dorsal CNS patterning. However, knowledge remains limited regarding the molecular mechanism regulating roof plate cell fate and function. Loss of function of Wnt/ß-catenin signaling in the roof plate or dorsal neural folds can prevent neural tube closure, neural crest cell production, and dorsal CNS patterning. In this study, we carried out conditional activation of ß-catenin in the dorsal neural folds of mice and found dramatic cell fate alterations, which include anteriorly expressed Cdx2 of the caudal-type homeobox gene in the dorsal neural tube, swapped expression patterns of the roof plate markers Bmp6 and Lmx1a with their respective homologues Bmp4 and Lmx1b, ectopically expressed Msx1 of the neural crest inducer in the migratory stream and dorsal root ganglia, and completely abolished expression of the neural crest cell differentiation marker Sox10 and the dorsal spinal interneuron precursor marker Atoh1. Our in vitro studies using a neural crest cell line demonstrate that the full-length ß-catenin, not the signaling defective ß-catenin significantly upregulates Msx1 and represses Sox10 and Atoh1 expression, which can be rescued by Msx1 knockdown, supporting an indirectly repressive role of the stabilized ß-catenin acting through Msx1 on Sox10 and Atoh1 expression. Intriguingly, Wnt1 and Wnt3a are drastically abolished in the roof plate of the ß-catenin stabilized embryos. However, Msx1 knockdown does not upregulate or rescue Wnt1 and Wnt3a expression when they are repressed by ß-catenin in vitro. Further in vitro studies reveal a direct repression of Wnt1 and Wnt3a by ß-catenin transcriptional complex, implicating a novel negative feedback loop of Wnt/ß-catenin signaling pathway at the ligand level. Together, these results suggest that constitutively stabilized ß-catenin triggers dual repression modes to prevent specification and differentiation of multiple cell lineages derived from the dorsal neural tube, but it may not prevent the presumptively undifferentiated Msx1-expressing neural crest cell precursors migrating out the dorsal neural tube to form the dorsal root ganglia, and that a finely tuned Wnt/ß-catenin signaling level is crucial for proper cell fate determination and functional modulation of the dorsal neural tube and its derivatives.
