Human liver tissue-derived organoids recapitulate key hepatic phenotypes but are commonly maintained under static conditions, whereas microfluidic organ-on-chip systems provide controllable perfusion and mass transport. Scalable integration of human liver tissue-derived organoids into a perfused, human-relevant Liver-on-Chip remains limited. We combined healthy human liver tissue-derived organoids with a high-throughput three-lane OrganoPlate microfluidic format to establish a perfused organoid Liver-on-Chip (HepLoC) featuring 3D luminal tubules under continuous flow. After hepatocyte-directed differentiation under perfusion, bioengineered HepLoC formed mature hepatocyte-like architectures with increased mature hepatocyte marker proteins, enrichment of hepatic transcriptomic signatures, and functional bile canaliculi. As a proof-of-concept for drug-induced liver injury, troglitazone induced dose-dependent hepatocyte injury accompanied by tight-junction disruption, MRP2 mislocalization, and impaired bile acid export, recapitulating key features of cholestatic liver injury. To model metabolic liver disease, free fatty acids triggered lipid droplet accumulation, increased triglycerides and reactive oxygen species, and upregulated lipogenic and inflammatory genes while largely preserving viability, consistent with early-stage metabolic dysfunction-associated fatty liver disease. The high-throughput HepLoC format further enabled parallel testing of reference hepatotoxic drugs and curcumin liposomes by reduced lipid accumulation in fatty-acid-treated HepLoC with minimal hepatotoxicity. Our perfused, organoid-based microfluidic Liver-on-Chip recapitulates essential human liver structure and function and enables integrated, parallel evaluation of hepatotoxicity and optimization of nanotherapeutic strategies, which deciphers the mechanisms of liver diseases, bridging the gap between preclinical research and clinical translation.
SARM1 (sterile α and TIR motif-containing protein-1) is an NADase enzyme that serves as the central executioner of Wallerian axon degeneration. Given this, SARM1 is of high interest as a candidate therapeutic target, and SARM1 inhibitors are currently in clinical trials for treatment of neurodegeneration. Beyond neuroscience, emerging studies reveal that SARM1 may also drive aspects of bone fragility, liver pathology, adipose expansion, and insulin resistance in metabolic disease. However, we lack methods to quantify SARM1 activation in humans to better define patients at high risk of SARM1-mediated tissue damage. Unlike neurons, peripheral blood mononuclear cells (PBMCs) represent an easily accessible population for clinical screening. While SARM1 gene expression has been identified in PBMCs, it is less known whether functional SARM1 NADase is present. We hypothesized that by pairing activators and inhibitors of SARM1 with analysis of downstream changes in cellular metabolites, we could identify and quantify both basal SARM1 activity and the SARM1 activation potential of human PBMCs. Our results reveal that SARM1 agonist pyrinuron, also known as Vacor, activates a dose-dependent increase in cAPDR and the cADPR:ADPR ratio that is arrested when paired with SARM1 inhibitor DSRM-3716. Changes in secondary metabolites including NAD+, NMN, NaMN, ATP, AMP, IMP, inosine, and succinyl adenosine were also characterized and used to generate a working model of PBMC SARM1 activation. Overall, these findings demonstrate that human PBMCs have detectable SARM1 activation potential and could be leveraged as a clinical readout of SARM1 expression and activity across diverse disease contexts.
Ferroptosis-a regulated, iron-dependent cell death driven by lipid peroxidation-has been implicated in vascular pathology, yet how endoplasmic reticulum (ER) stress couples to ferroptotic execution in vascular smooth muscle cells (VSMCs) remains unclear. We investigated whether the PERK-EIF2α-ATF4-CHAC1 signalling axis links ER stress to ferroptosis in human aortic SMCs. Human aortic smooth muscle cells (HASMCs) and primary aortic smooth muscle cells (AoSMCs) were exposed to erastin (± ferrostatin-1), with pharmacologic modulation using buthionine sulfoximine (BSO; glutamate-cysteine ligase inhibitor) and salubrinal (selective EIF2α dephosphorylation inhibitor). Viability (CCK-8), ER-stress markers (p-PERK, p-EIF2α, GRP78, ATF4, CHAC1) and ferroptosis effectors (GPX4, ACSL4, ALOX15) were quantified by Western blotting and densitometry. Immunofluorescence assessed GPX4 and p-EIF2α/CHAC1 colocalization. Lipid peroxidation was evaluated by C11-BODIPY 581/591 imaging and malondialdehyde (MDA) content; labile Fe2+ was measured by calcein-AM quenching; apoptosis was examined by TUNEL. Statistics used t-tests or one-way ANOVA. Erastin reduced viability and activated the EIF2α-ATF4-CHAC1 pathway in both HASMCs and AoSMCs, as evidenced by increased p-EIF2α/ATF4/CHAC1 and decreased GPX4, with concomitant upregulation of ACSL4 and ALOX15. GPX4 immunofluorescence declined, while C11-BODIPY and MDA indicated robust lipid peroxidation, and calcein-AM revealed expansion of the labile Fe2+ pool; ferrostatin-1 mitigated these changes. BSO further amplified p-PERK→p-EIF2α → ATF4 → CHAC1 signalling and deepened GPX4 loss, whereas salubrinal sustained EIF2α phosphorylation, augmented ATF4/CHAC1, and further depressed GPX4. Across conditions, effects were consistent in both cell types and reached statistical significance (most p < 0.05 to < 0.01). ER-stress signalling via PERK-EIF2α-ATF4-CHAC1 constitutes a proximal driver of ferroptosis in human aortic SMCs by eroding glutathione tone and disabling GPX4, thereby promoting lipid peroxidation in an iron-rich milieu. Pharmacologic tuning of EIF2α phosphorylation and glutathione biosynthesis modulates ferroptotic susceptibility, nominating EIF2α-ATF4-CHAC1, GPX4/GSH homeostasis and iron handling as actionable nodes to preserve VSMC integrity in oxidizing vascular environments.
While formalin-fixed paraffin-embedded (FFPE) samples are invaluable for human non-Hodgkin B-cell lymphoma translational research, effective methods for spatial profiling of chromatin accessibility and histone modifications in these tissues remain limited. Here, we introduce epi-Patho-DBiT, a platform that combines reverse crosslinking of FFPE tissues with spatially resolved assays for transposase-accessible chromatin using sequencing (spatial-FFPE-ATAC) or cleavage under targets and tagmentation (spatial-FFPE-CUT&Tag). Using spatial-FFPE-ATAC, we map epigenetic landscapes in mucosa-associated lymphoid tissue and follicular lymphoma, identifying chromatin variants linked to B-cell malignancy and resolving tumor karyotypes. Mitotic age inference reveals spatial tumor dynamics and uncovers cholesterol-mediated cell proliferation. Furthermore, spatial-FFPE-CUT&Tag elucidates genomic alterations during transformation of follicular lymphoma into diffuse large B-cell lymphoma and identifies DIP2C with dysregulated H3K4me3 and H3K27me3 levels. Unexpectedly, we observe elevated H3K27me3 occupancy at a chromosome 2 locus containing tumor-promoting genes, attributed to copy number amplification and thereby upregulation in transformed diffuse large B-cell lymphoma.
Two isoforms of the 90-kDa heat shock protein (Hsp90), stress-inducible Hsp90α and constitutively expressed Hsp90β, function in mammalian cells as molecular chaperones that promote the folding of specific client proteins involved in essential cellular processes and regulatory pathways. A number of Hsp90 client proteins take part in cancer progression, and the inhibition of Hsp90 induces the degradation of oncogenic client proteins and cancer cell death. Hsp90 inhibitors specific for individual Hsp90 isoforms have a significant potential for the development of anticancer therapeutics due to reduced toxicity. Cells with knocked-out genes encoding Hsp90 isoforms represent excellent cellular models to investigate the rearrangement of the cell chaperone machinery in response to the suppression/loss of the Hsp90 isoforms. Recently, we have shown that the knockout of the HSP90AA1 gene encoding Hsp90α in human fibrosarcoma HT1080 cells does not affect basic cellular processes in normal and stressful conditions, which suggests an adaptation of the cell chaperone machinery to the loss of Hsp90α. Here, we demonstrated that the lack of Hsp90α in HT1080 cells leads to an up-regulation of the constitutively expressed Hsp90β and several important Hsp90 co-chaperones (Aha1, Hop, and others). The expression of the major chaperones of the Hsp70 machinery (Hsp70-1, Hsp70-2, Hsc70) was also significantly induced. The components of the prefoldin-chaperonin folding arm and PFDL, R2TP, and R2SP complexes, as well as the major mitochondrial chaperones, were also largely up-regulated in Hsp90α-KO cells, while the expression of ER-resident chaperones/co-chaperones was either repressed or did not change. We demonstrated here for the first time an adaptation of the cell chaperone machinery to the loss of the Hsp90α chaperone, which may be important for understanding the molecular mechanisms of action of Hsp90α-specific inhibitors and elaborating new therapy strategies in combating cancer, including the combination of Hsp90α-targeted therapy.
Therapeutic resistance is a major cause of treatment failure in glioblastoma (GBM), highlighting the need for physiologically relevant models to identify actionable resistance mechanisms. While two-dimensional (2D) cultures are widely used for target discovery, they poorly represent the tumor microenvironment. In contrast, three-dimensional (3D) spheroid cultures better recapitulate spatial heterogeneity, hypoxic gradients, and stress-adaptive signaling observed in tumors. We applied an integrated 2D-3D quantitative proteomic approach to identify microenvironment-dependent regulators of chemoresistance in GBM. Proteomic profiling was performed in U87MG and U251MG cells grown as 2D monolayers or 3D spheroids. Differentially expressed proteins were validated by quantitative RT-PCR, and functional studies were conducted using genetic depletion followed by assessment of temozolomide (TMZ) sensitivity. Comparative analysis identified 13 proteins consistently differentially expressed between 2D and 3D cultures: NDUFB5, RNGTT, MLK4, SYN1, DDX5, EIF2AK2, ITGA1, ZNF33B, ZNF343, WDR19, JPH3, CCT8L2, and FNDC3A. Among these, Mixed Lineage Kinase 4 (MLK4) showed strong and reproducible upregulation in 3D spheroids in both GBM cell lines. Genetic depletion of MLK4 significantly increased TMZ sensitivity without affecting basal cell viability, suggesting a specific role in therapy response. Notably, MLK4 expression was induced only under 3D conditions. MLK4 functions as a microenvironment-responsive regulator of chemoresistance in GBM. These findings demonstrate that 3D culture systems reveal clinically relevant resistance pathways not detectable in conventional 2D models and highlight 3D proteomic profiling as a powerful strategy for identifying therapeutically actionable targets.
Pediatric thyroid nodules are uncommon but have higher malignancy risk than adult nodules. The 2023 TBSRTC updated ROM estimates and expanded pediatric guidance, but data remain limited. We reviewed 63 thyroid FNAs from patients aged 21 years or younger at a tertiary center (January 2023-October 2025). Cases were classified according to the 2023 TBSRTC. Observed ROM was calculated in resected nodules with exact 95% CIs. Bethesda III nodules were subclassified as AUS with nuclear atypia or AUS-other. Diagnoses were Bethesda I in 5/63 (7.9%), Bethesda II in 32/63 (50.8%), Bethesda III in 3/63 (4.8%), Bethesda IV in 10/63 (15.9%), Bethesda V in 13/63 (20.6%), and Bethesda VI in 0/63 (0%). Histologic follow-up was available for 36 nodules. Observed ROM among resected nodules was 0/1 (0.0%) for Bethesda I, 1/11 (9.1%) for Bethesda II, 2/3 (66.7%) for Bethesda III, 4/8 (50.0%) for Bethesda IV, and 12/13 (92.3%) for Bethesda V. The two malignant Bethesda III cases were AUS with nuclear atypia; the AUS-other case was benign. Overall malignant yield was 19/36 (52.8%). The 2023 TBSRTC was applicable in this pediatric cohort. Bethesda II, IV, and V ROM estimates were broadly consistent with published pediatric data, whereas Bethesda III was imprecise because only three nodules were resected. Larger multicenter studies with standardized follow-up and explicit AUS subclassification are needed.
Synaptic function and plasticity depend on the precise control of protein abundance and turnover, governed by the balance of synthesis and degradation. This review examines the regulatory mechanisms that maintain synaptic protein stability, focusing on the Ubiquitin-Proteasome System (UPS), autophagy-lysosomal pathways, and related proteolytic systems. We detail how key enzymes, including E3 ligases such as Nedd4-1, Mdm2, and Parkin, and deubiquitinating enzymes like USP46 and USP8, dynamically regulate the degradation of critical synaptic components from AMPA and NMDA receptors to scaffolds like PSD-95 and SHANK3. We further explore how autophagy, including chaperone-mediated and activity-dependent forms, contributes to synaptic remodeling and quality control. Crucially, dysfunction of synaptic degradation pathways is a common thread in neurodevelopmental and neurodegenerative disorders. We summarize evidence linking proteostatic malfunction to the pathogenesis of Alzheimer's disease (through impaired clearance of Aβ and tau), Parkinson's disease (via α-synuclein turnover), epilepsy, autism spectrum disorder, and ischemic injury. The review highlights how genetic mutations in degradation machinery or their synaptic targets converge to disrupt synaptic integrity and neural circuit function. By integrating findings from basic neurobiology and disease models, this review underscores the central importance of synaptic proteostasis and aims to identify critical regulatory molecules that retain potentials for diagnostic biomarkers and therapeutic targets for neurological disease.
Although Baker's cysts are commonly associated with meniscal tears, the role of specific tear morphologies and anatomic locations in cyst presence remains unclear. This study aimed to evaluate the associations between meniscal tear patterns, tear locations, and Baker's cysts and to explore whether these tear characteristics were also associated with concomitant chondral lesions. In this retrospective cohort study, patients who underwent knee arthroscopy at a single institution were evaluated and categorized according to the presence or absence of a Baker's cyst on MRI. Arthroscopic video recordings were reviewed to identify meniscal tear morphology and tear location based on the Cooper classification, as well as the presence of chondral lesions. Separate multivariate logistic regression models were constructed for tear morphology and tear location to account for collinearity among meniscal variables. Of 353 patients (mean age 35.5 ± 13.4 years), 77 (21.8%) had a Baker's cyst. In the multivariate tear morphology model, horizontal, radial, and complex medial meniscal tears were significantly associated with Baker's cysts (OR: 7.321, 95% CI: 2.921-18.349, p < 0.001; OR: 3.380, 95% CI: 1.136-10.056, p = 0.039; and OR: 4.000, 95% CI: 1.907-8.394, p < 0.001, respectively). In the multivariate location model, tears involving Cooper zones A2, A3, and B3 were also significantly associated with Baker's cysts (OR: 2.956, 95% CI: 1.133-7.713, p = 0.027; OR: 3.978, 95% CI: 1.838-8.606, p < 0.001; and OR: 7.070, 95% CI: 2.878-17.371, p < 0.001, respectively). Chondral lesions remained independently associated with Baker's cysts in both models. In univariate analyses, these tear types and locations were also associated with chondral lesions, although these associations were not maintained after multivariate adjustment. In this retrospective cohort, horizontal, radial, and complex medial meniscal tears, particularly those involving Cooper zones A2, A3, and B3, were associated with the presence of Baker's cysts. These findings expand the current literature by providing a more detailed description of the relationship between meniscal tear morphology, tear location, and Baker's cysts, and may serve as a basis for future studies investigating the underlying mechanisms.
Placental histopathology provides important insights into maternal and fetal health, yet the organ's spatial heterogeneity poses significant challenges for objective and reproducible histological analysis. Systematic assessment of cellular and structural composition across placental slides remains limited by the scale and subjectivity of manual evaluation. Quantitative approaches are therefore needed to characterise placental responses to injury beyond visually apparent lesions. We applied the Histology Analysis Pipeline.PY (HAPPY), a biologically inspired hierarchical deep learning framework for quantitative single-cell-resolution analysis of Haematoxylin and Eosin (H&E) slides, to 130 placental parenchyma slides from 62 singleton full-term live births. The dataset included healthy normal controls and four common placental lesion types: infarction, perivillous fibrin, avascular villi, and intervillous thrombosis. Cell-type and tissue-structure compositions were quantified, and slide-level deviation from a healthy reference was assessed using compositional data analysis. Placental slides with lesions exhibited significant cellular composition differences compared with healthy controls, including increased extravillous trophoblast and leukocyte densities and decreased Hofbauer cell densities. These cellular changes were accompanied by tissue-level alterations, particularly increased fibrin deposition and changes in villous structure. Compositional deviation increased with infarction size but not with other lesion types. Notably, compositional differences were also detected in slides without an apparent lesion from placentas with lesion(s) elsewhere, indicating organ-wide responses extending beyond focal pathology. Quantitative deep phenotyping reveals widespread cellular and structural changes associated with placental lesions, including effects not evident on routine histological assessment. These findings demonstrate the potential of AI-based digital histology to complement conventional placental pathology in research and clinical settings.
Animal models are crucial for mechanistic studies and therapeutic development of human diseases. At present, the etiology of interstitial cystitis/bladder pain syndrome (IC/BPS), a chronic disease of the urinary bladder, remains undefined. Therefore, numerous theories of pathogenesis have been proposed, and various animal models have been developed based on these theories. This enigmatic human disease can be categorized into two subtypes: Hunner-type IC (HIC) and bladder pain syndrome (BPS). These two subtypes of IC/BPS have different pathological mechanisms, but their clinical symptoms overlap. Recent evidence indicates that HIC is an immune-mediated inflammatory disease of the urinary bladder, while BPS is a minimally inflamed bladder condition comprising various clinical phenotypes. Furthermore, increasing evidence suggests that autoimmunity may play a significant role in IC/BPS, particularly in HIC. Today, the rodent models of experimental autoimmune cystitis (EAC) are being used in HIC research. This article provides an overview of immune-mediated inflammation and autoimmunity in IC/BPS, as well as EAC models that can be used for HIC research, with a focus on the URO-OVA model, a novel transgenic EAC model that effectively mimics HIC. The URO-OVA model develops chronic bladder inflammation, pelvic/bladder pain, and voiding dysfunction seen in human HIC patients. It responds to treatment with dimethyl sulfoxide (DMSO) and specific inhibitors, such as Toll-like receptor (TLR)4, mitogen-activated protein (MAP) kinase, and interferon (IFN)-γ inhibitors. The URO-OVA model is stable and reproducible, providing a unique EAC model for HIC research that incorporates immune/autoimmune components in its pathophysiology.
Cytokine-mediated cross-talk between immune cells and fibroblasts is a driver of excessive ECM accumulation during fibrosis. In this study, we used a 3D in vitro model of a connective tissue to discern the roles of three pro-inflammatory cytokines; TNF-α, IL-18 and IL-1β, alone, and in combination with TGF-β1 to simulate the fibrotic environment. Ring-shaped tissues were formed by seeding human fibroblasts into circular molds of agarose, wherein the cells self-assembled, formed a 3D tissue and synthesized de novo a collagen-rich ECM. Cytokine treated tissues were analyzed at days 7 and 14 by histology and measured for thickness, collagen, DNA and strength and stiffness by tensile testing. Despite their pro-inflammatory nature, none of the cytokines increased collagen alone or in combination with TGF-β1. TNF-α and IL-1β reduced collagen, tissue strength and stiffness, and altered tissue morphology. When combined with TGF-β1, TNF-α and IL-1β counteracted TGF-β1-mediated increases in collagen, strength, and stiffness. In contrast, IL-18 had minimal effects alone or when combined with TGF-β1. These data suggest that IL-18 has no effect on fibroblast activation, whereas TNF-α and IL-1β may modulate TGF-β1's effects. This 3D model of a human collagen-rich tissue can help define cytokine-mediated cross-talk between immune cells and fibroblasts.
Duchenne muscular dystrophy (DMD) is a severe X-linked disorder marked by progressive muscle degeneration and regeneration, inflammation and fibrosis. Cellular senescence has emerged as a potential driver of chronic muscle damage, yet its temporal dynamics and therapeutic relevance remain unclear. We analyzed senescent cell burden in skeletal and cardiac muscles of the DBA/2-mdx mouse model, which closely mimics features of human DMD. The senolytic combination of dasatinib and quercetin (D + Q) was administered during early or late disease phases to evaluate the impact of senescent cell clearance. Skeletal muscle strength was measured by grip strength and ex vivo force assays, while cardiac function was assessed by echocardiography. Fibrosis and senescence markers were quantified histologically, and transcriptional changes associated with senolysis were identified using bulk RNA sequencing (RNA-seq). In skeletal muscle, senescent cells appear and peak during early stages of disease progression (3-5 months), coinciding with high degeneration and regeneration activity, and then decline with age as fibrosis increases. In contrast, in the heart, senescent cells emerge at late stages of disease progression (around 12 months), correlating with heart fibrogenesis. Notably, senolytic intervention in the DBA/2-mdx mice promotes a regenerative and antifibrotic gene signature in both tissues. However, the timing of senolytic therapy determines its efficacy: early treatment with D + Q reduces senescent cell burden, decreases fibrosis, and improves fiber size and contractile performance in skeletal muscle, while later treatment reduces cardiac senescence and fibrosis but does not improve skeletal muscle pathology. Cellular senescence is a dynamic and targetable feature in DMD, with tissue- and age-specific patterns. It represents a potential modifiable therapeutic target, and temporally optimized senolytic strategies could serve as effective adjuncts to current and emerging DMD treatments.
Colorectal cancer (CRC) represents a prevalent and life-threatening malignancy, posing a significant global health challenge. Transcribed ultraconserved regions (T-UCRs), a specific category of long non-coding RNAs (lncRNAs) encoded within the human genome, have been demonstrated to play significant roles in the pathogenesis of multiple cancer types. However, their pathological role in CRC remains largely unexplored. In this study, we investigate two closely spaced T-UCRs, uc.263 and uc.264, located on chromosome 9, both of which are significantly upregulated in CRC tissues. Our findings further reveal that these two T-UCRs originate from a shared precursor RNA, designated as uc.263/264 in CRC cells. The uc.263/264 has been demonstrated to enhance cellular proliferation, growth, migration, and invasion, while simultaneously regulating the cell cycle and apoptosis. Mechanistically, uc.263/264 interacts with heterogeneous nuclear ribonucleoprotein K (hnRNPK), enhancing its protein stability and resulting in increased hnRNPK expression. Furthermore, uc.263/264 activates the Wnt signaling pathway, a process mediated by hnRNPK. Clinically, both uc.263/264 and hnRNPK are overexpressed in CRC patient samples, exhibiting a positive correlation in their expression levels. This suggests a functional regulatory axis between uc.263/264 and hnRNPK in CRC pathology. Collectively, our findings demonstrate that uc.263/264 can interact with the hnRNPK protein and upregulate its expression, thereby activating the Wnt signaling pathway and promoting the progression of CRC.
Dystrophinopathies are caused by pathogenic variants in the DMD gene, resulting in partial (Becker) or complete loss (Duchenne) of dystrophin. Becker (BMD) and Duchenne muscular dystrophy (DMD) are characterized by progressive muscle wasting, fatty replacement, fibrosis, and loss of function. To study histopathological changes, we used Visium spatial transcriptomics to profile skeletal muscle biopsies of patients affected by dystrophinopathy (n = 8) and healthy controls (n = 4). We estimated the proportion of cell types and their spatial localization across samples applying a deconvolution strategy using previously published single-nucleus RNA-sequencing data. We identified genes enriched in fat patches and cell types such as fibroadipogenic progenitors (FAPs) in areas of active pathology. Using expression data of ligand-receptor pairs, we highlight cell-cell communications leading to fibrotic and adipogenic lesions. Finally, analysis of gene expression gradients in areas of adjacent muscle and fat, allowed the identification of genes associated with muscle areas committed to becoming fat. © 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Intervertebral disc degeneration (IDD), a leading cause of low back pain, involves progressive dysfunction of nucleus pulposus (NP) cells and extracellular matrix degradation. The pathological mechanisms underlying IDD remain complex and lack comprehensive elucidation. This study identifies the RNA-binding protein TDP43 as a central driver of IDD pathogenesis through analysis of human clinical specimens and rodent models. We demonstrate that TDP43 expression escalates proportionally with disc degeneration severity and aberrantly accumulates in the mitochondria of degenerative NP cells. This mitochondrial mislocalization triggers nuclear pore complex impairment, mitochondrial membrane potential collapse, and irreversible cellular senescence. Critically, TDP43 is secreted within mitochondrial-derived vesicles, which function as intercellular mediators that propagate pro-inflammatory cytokines and senescence phenotypes to neighboring NP cells. Both genetic and pharmacological inhibition of vesicular TDP43 effectively attenuated mitochondrial dysfunction and reduced cellular senescence and ultimately decelerated IDD progression in vivo and in vitro. Our findings establish TDP43-loaded mitochondrial-derived vesicles as novel mediators of intercellular pathology and nominate TDP43 as a therapeutic target for IDD intervention.
Defective clearance of apoptotic hepatocytes contributes to inflammation and progression of alcohol-associated liver disease (ALD), but the mechanisms regulating macrophage efferocytosis during alcohol exposure remain unclear. We investigated whether fatty acid synthase (FASN)-dependent lipid metabolism controls hepatic macrophage efferocytosis in ALD. Human liver tissues from patients with alcohol-related cirrhosis (AC) and controls (n = 18/group), together with experimental ALD mouse models (n = 6/group), were analyzed for hepatocyte apoptosis and hepatic macrophage alterations. Transcriptomic profiling (n = 3/group), pharmacological inhibition, and myeloid- or Kupffer cell-specific Fasn knockout mice (n = 6/group) were used to define the role and mechanism of FASN-mediated lipogenesis in macrophage efferocytosis. In patients with AC, hepatocyte apoptosis was markedly increased compared with controls (p < 0.0001), accompanied by increased accumulation of CD68-positive inflammatory macrophages (p < 0.001). In experimental ALD mice, hepatocyte apoptosis and monocyte-derived macrophage infiltration were also significantly increased (p < 0.0001). Mechanistically, ethanol impaired macrophage efferocytosis by more than 80% (p < 0.01). This was associated with inhibition of the PI3K/AKT/SREBP1 pathway, reduced FASN expression, and suppressed de novo lipogenesis. Reduced FASN expression decreased NRF2 activity and impaired TREM2 transcription, resulting in defective clearance of apoptotic cells. TREM2-positive hepatic macrophages were markedly reduced in both human AC and murine ALD (p < 0.0001). Consistently, Kupffer cell-specific Fasn deletion significantly aggravated hepatocyte apoptosis and liver injury in vivo (p < 0.01). Alcohol impairs macrophage efferocytosis by suppressing the PI3K/AKT/SREBP1-FASN-NRF2-TREM2 axis. Disruption of this lipogenic program promotes hepatocyte apoptosis and liver inflammation in ALD. Alcohol-associated liver disease is characterized by hepatocyte death and ongoing inflammation, but the mechanisms that connect these processes to macrophage efferocytosis remain poorly understood. Our research revealed that ethanol suppresses FASN-dependent de novo lipogenesis and downstream NRF2-TREM2 signaling in hepatic macrophages. Impairment of lipogenesis compromises efferocytosis, leading to an accumulation of apoptotic hepatocytes and increased monocyte infiltration. These findings underscore the potential of targeting macrophage lipid metabolism as a therapeutic strategy in ALD. However, further translational validation is needed before clinical application.
Acute ischemic stroke (AIS) is one of the leading causes of death and disabilities, and as such, it is of utmost importance to identify novel treatment options. Remote ischemic conditioning (RIC) is a promising non-invasive treatment that is thought to activate the body's own protective mechanisms against damaging ischemia. Here, we study the transcriptomic impact of microRNAs (miRNAs) that are upregulated by RIC. Using RNA sequencing, we investigated the transcriptional changes in human brain microvascular endothelial cells (HBMECs) transfected with four selected RIC-upregulated miRNAs (RIC-miRNAs), miR-16-5p, miR-144-3p, miR-182-5p, and miR-451a, under oxygen and glucose deprivation (OGD) and reoxygenation-mimicking the initial stages of AIS. Pronounced transcriptional changes were present after RIC-miRNA transfection, with 149 unique downregulated and 212 upregulated differentially expressed genes in HBMECs after OGD and RIC-miRNA transfection compared to all other conditions. These genes were involved in pathways of energy metabolism and cell cycle regulation. Our study suggests that the selected RIC-miRNAs regulate pathways that may facilitate endothelial cell survival, recovery, and remodeling events from ischemic damage, adding to the knowledge of the pathways affected by RIC during stroke.
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases with overlapping pathology. Mutations in CCNF, encoding the E3 ubiquitin ligase, Cyclin F, can cause ALS, FTD, or both, even within the same family. Most prior studies of CCNFS621G have relied on overexpression systems, potentially confounding outcomes through disruption of endogenous Cyclin F. Here, we generated the first knock-in mouse model of endogenous CcnfS621G using CRISPR/Cas9. Heterozygous and homozygous CcnfS621G mice showed no motor decline or neuronal loss after 18 months, however immunohistochemistry revealed increased hippocampal astrocyte ramification, with sex-, age, and subfield-dependent effects. These data indicate that endogenous CcnfS621G may prime early astrocyte alterations in the absence of overt neurodegeneration. Similar astrocyte morphological changes were observed in canonically affected regions of sporadic ALS and FTD-ALS patients post mortem, as well as in CCNFS621G iPSC-derived astrocytes following inflammatory stimulation. Proteomics on Ccnf mice identified early dysregulation of pathways related to translation, mitochondrial function, cytoskeletal remodelling, synaptic transmission and neuroinflammation. Correspondingly, CCNFS621G iPSC-derived astrocytes displayed impaired mitochondrial membrane potential and altered network morphology under both basal and inflammatory stimuli. As altered neuronal excitability is a hallmark of ALS, we examined astrocyte-driven changes to neuronal excitability. CCNFS621G iPSC-derived motor neurons cultured alone were hyperexcitable, firing more action potentials than isogenic controls. Remarkably, co-culture with CCNFS621G astrocytes, but not isogenic control astrocytes, abolished repetitive firing, increased the proportion of neurons unable to generate action potentials, and reduced voltage-gated sodium currents in CCNFS621G and isogenic control neurons. Together, these findings identify astrocyte alterations as an early feature of CCNFS621G-mediated disease, in the absence of neuronal loss. Moreover, the combination of astrocytic mitochondrial dysfunction and the ability of CCNFS621G astrocytes to suppress repetitive neuronal firing suggests a critical astrocyte-driven non-cell autonomous mechanism that may contribute to an oligogenic role for CCNF in ALS/FTD pathogenesis.
Estradiol (E2), a sex steroid hormone molecule, plays a key role in regulating the actin and shape dynamics of cells in a multitude of normal and pathophysiological conditions. While cytoskeletal rearrangements, membrane dynamics, and cellular protrusions are intimately involved in cell motility and invasiveness, little is known about the impact of E2 on these processes in estrogen-dependent epithelial cells. In this study, we quantified the impact of E2 on epithelial cell shape and actin dynamics. 12Z human endometriotic epithelial cells were transfected with LifeAct-GFP and observed with lattice lightsheet microscopy, a new imaging technique fast enough to capture 3D dynamics on second timescales. E2, when applied for 24 h, significantly decreased cell circularity, solidity, and rate of change of circularity, indicating a transition to a more elongated and less variable morphology. 24-h E2 treatment also induced the formation of large membrane protrusions reminiscent of invadopodia and led to a more disordered flow of actin within those protrusions. However, these effects were not seen after 15 min of E2 treatment, suggesting that longer-term signaling is required to drive these structural changes. Together, these results suggest that E2 modulates actin polymerization and membrane protrusion dynamics in endometriotic epithelial cells and may prime them for cell invasion. This work highlights a role for hormonal signaling in mediating cytoskeletal plasticity and migratory cell phenotypes.