Cells

PurposeFuchs endothelial corneal dystrophy (FECD) is a progressive disease, causing premature death of corneal endothelial cells (CECs). Iron-dependent lipid peroxidation and ferroptosis mediate cell death in FECD. We aimed to determine whether FECD progression is mediated by derangements in ferritinophagy - a form of autophagy that degrades ferritin to release labile ferrous iron - and whether ultraviolet A (UVA) exposure drives FECD progression by activating ferritinophagy. MethodsEndothelium-Descemet membrane (EDM) tissues were collected from patients with end-stage FECD undergoing endothelial keratoplasty and from healthy age-matched donor corneas. Separately, immortalized FECD and healthy control CEC lines were cultured. Cellular levels of NCOA4 production and LC3 activation, both markers of ferritinophagy, were quantified using western blotting and PCR. UVA-exposed immortalized cells were plated on coverslips, stained for immunohistochemistry (IHC), and analyzed using confocal microscopy. Corneal endothelial peels were stained and analyzed using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). ResultsSurgically explanted FECD CECs showed significantly increased levels of NCOA4 compared to healthy controls. LC3 activation was increased in FECD immortalized CECs; UV exposure further increased LC3 activation. Additionally, UVA exposure showed trends of increased expression of NCOA4 in immortalized FECD and healthy CECs. On IHC of FECD surgical explant tissue, ferritin was decreased markedly, NCOA4 localized in a dramatic punctate pattern, and both ferritin and LC3 localized within cell nuclei. Spectrometry images showed higher iron levels correlating with areas of higher FECD disease burden. ConclusionsOur results demonstrate ferritinophagy in FECD indicated by the increase of NCOA4 and LC3 ferritinophagy markers in FECD patient and cell culture models. Our finding that UVA activates ferritinophagy implicates this mechanism in UVA-mediated FECD progression. Altogether, aberrant iron dysregulation associated with FECD and ferroptosis may be mediated by ferritinophagy, providing a biomarker to assess disease severity as well as a potential target for future medical therapeutics.

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss in older adults. It is characterised by early retinal pigment epithelium (RPE) dysfunction followed by progressive photoreceptor degeneration. Cigarette smoking is a major environmental risk factor for AMD, and hydroquinone (HQ), a redox-active cigarette smoke component, induces oxidative stress and apoptosis in RPE cells. To analyse how RPE stress contributes to photoreceptor degeneration, we employed a retinal co-culture model composed of human induced pluripotent stem cell-derived RPE (iPSC-RPE) cells in conjunction with porcine neuroretina explants. Exposure to HQ induced oxidative stress in iPSC-RPE cells as well as retinal photoreceptors (RPR), resulting in apoptosis, executed at least in part by caspase activation. Concomitantly, HQ caused endoplasmic reticulum (ER) stress (ERAD) in RPR followed by their degeneration, evidenced by reduced outer nuclear layer (ONL) rows and shortened RPR outer segments (OS). Based on earlier results, which suggest a perturbation of proteostasis due to HQ, we tested whether ML240, a bona fide inhibitor of valosin-containing protein (VCP), would influence the degree of degenerative activities. ML240 did not prevent HQ-induced apoptosis in iPSC-RPE cells. However, it significantly preserved photoreceptor integrity, retaining OS length and cone density in HQ-stressed co-cultures. Proteomic analysis suggested that ML240 reshapes stress response patterns of the HQ-exposed neuroretina, as evidenced by a reduction in ERAD-associated markers, increased levels of antioxidant response proteins, and the preservation of cytochrome c enrichment in photoreceptor inner segments, which indicates improved mitochondrial integrity consistent with the observed preservation of photoreceptor structure. Together, these findings establish the iPSC-RPE/neuroretina co-culture as a platform to analyse pathophysiological features of AMD, dissect cell type-specific retinal responses to environmental stress and test neuroprotective pharmacological approaches to protect photoreceptors in oxidative stress-associated retinal degeneration.

Purpose: Oxidative stress is thought to contribute to the development of age-related cataracts (ARCs), but the mechanisms by which oxidative damage leads to the opacification of the lens remain unclear. Previous studies suggest that oxidative stress can disrupt lens proteostasis. Therefore, it was hypothesized that ARCs arise from proteomic changes driven by an age-associated decline in oxidative stress defenses that interact with the lens proteostatic state. To test this hypothesis, proteomic analyses of lenses exposed to oxidative stress were performed to examine oxidative and proteostatic stress responses in vivo. Methods: Cataract formation was induced by injecting hydrogen peroxide into the aqueous humor of adult zebrafish. nrf2fh318/fh318 zebrafish were used to model the reduced oxidative stress protection observed in aged human lenses, while cryaba-/- zebrafish were used to model impaired lens proteostasis. Resulting opacities in WT, cryaba-/-, nrf2fh318/fh318, and cryaba-/-; nrf2fh318/fh318 lenses were quantified and proteomic changes in the cortex were analyzed using data independent acquisition Parallel Accumulation Serial Fragmentation mass spectrometry. Results: Hydrogen peroxide treatment induced the formation of cortical cataracts. Proteomic results showed that, dependent on genotype and day, oxidative stress activates the unfolded and mitochondrial unfolded protein responses. Additional changes were also observed in energy metabolism, Ca2+ homeostasis, protein degradation, and cytoskeletal and extracellular matrix remodeling pathways. Conclusions: Treated zebrafish lenses successfully model ARC and mass spectrometry proteomics identified the unfolded and mitochondrial unfolded protein responses as potential therapeutic targets for ARC.

Human iPSC-derived brain organoids are revolutionizing tools to study layers biology, synergize disease modeling, and accelerate therapeutic discoveries that overcome obstacles in monolayer cell culture or animal models. The neurovascular unit including vasculature and microglia is critical for brain development, maintenance of synaptic plasticity and neural activity, and the high metabolic demands of long-term culture. We present a methodology to incorporate these important components during organoid generation and discuss potential approach, aiming consistent production of vascularized organoids for longitudinal study. We also demonstrate that this vascularized organoid is a versatile platform to model brain cancer and traumatic brain injury.

Traumatic optic neuropathy (TON) occurs due to direct or indirect injury to the optic nerve and is a significant cause of visual disability. So far, there is no effective treatment. The lack of understanding of the cellular mechanisms by which trauma induces inflammation and damage in retinal neurons is a critical knowledge gap in developing effective therapies. We have studied the role of the arginase 1 (A1) enzyme in this pathology. We have found previously that treatment with a long-acting form of human recombinant A1, pegylated A1 (PEG-A1) after optic nerve crush limits activation of retinal microglia and macrophages (M{Phi}) and reduces inflammation, thereby decreasing injury and protecting visual function. Here we report on studies designed to demonstrate the therapeutic efficacy of PEG-A1 in mouse models of direct and indirect TON and to elucidate the underlying mechanisms. We used ONC to model direct TON and sonication-induced trauma to the supraorbital rim to model indirect TON (SI-TON). At different times after injury, mice were treated with PEG-A1 which was delivered systemically by i.p. injection or locally by intravitreal injection. In order to assess the role of A1-induced activation of the ornithine/polyamine pathway in the protective effects of PEG-A1, some mice were treated with the ornithine decarboxylase (ODC) inhibitor, difluoromethylornithine (DFMO) immediately after the PEG-A1 treatment. Retinal function was determined by OptoMotry and electroretinography. Retinal injury and microglia/M{Phi} activation were assessed by immunofluorescence imaging. Expression of inflammatory cytokines was determined by Western blotting and quantitative RT PCR. Liquid chromatography mass spectrometry was used to analyze changes in arginase/ODC pathway metabolites. Results showed that PEG-A1 treatment improved neuronal survival and visual function whether delivered systemically or intravitreally. This neuroprotection was associated with decreased microglia/M{Phi} activation, decreased inflammatory cytokine expression, and increased formation of L-ornithine and putrescine. Furthermore, DFMO treatment blocked these effects, indicating that PEG-A1 limits retinal injury and preserves vision after ocular injury by activating ODC. ODC processes the arginase product L-ornithine to form polyamines which are known to promote reparative functions. Thus, PEG-A1 therapy offers a new strategy to limit trauma-induced vision loss and promote repair after TON.

ObjectiveThe complement regulatory protein CD59 has been shown to promote SNARE complex assembly, yet its interaction with vesicle-associated membrane protein 2 (VAMP2) remains poorly characterized. This study aims to identify the key domain in VAMP2 that mediates the CD59 interaction and to evaluate whether CD59 point mutations affect SNARE complex assembly. MethodsThe interaction between CD59 and VAMP2 was examined by immunofluorescence confocal microscopy and co-immunoprecipitation (co-IP). The effect of CD59 on SNARE complex assembly was assessed by co-expressing CD59 with the three core SNARE proteins (syntaxin-1, SNAP-25, and VAMP2) and detecting complex formation by western blotting. Four CD59 single-point mutants were generated and evaluated in SNARE assembly assays. AlphaFold3 was employed to predict the interaction between CD59 and individual VAMP2 domains (confidence threshold: ipTM + pTM [≥] 0.75). Truncated VAMP2 constructs were further characterized by molecular dynamics simulations and co-IP. Results(1) CD59 directly bound VAMP2 and promoted SNARE complex assembly without altering individual SNARE protein levels. (2) All four CD59 single-point mutants retained the ability to promote SNARE assembly at a level comparable to wild-type CD59, despite showing differential effects on binding stability in molecular dynamics simulations. (3) The proline-rich (P-rich) N-terminal domain of VAMP2 was identified as the key binding interface; its deletion abolished the CD59 interaction, whereas deletion of the SNARE motif did not. ConclusionCD59 promotes SNARE complex assembly through interaction with the P-rich N-terminal domain of VAMP2. The examined point mutations do not impair this function, suggesting that these sites may tolerate substitutions or that redundant contact residues maintain the interaction.

The emergence of bispecific antibodies (BsAbs) targeting T cells (CD3+) and tumor plasma B cells (BCMA+) has provided a new therapeutic option for patients with relapsed/refractory multiple myeloma cancer. However, responses to CD3xBCMA BsAb therapy remain heterogeneous, and treatment is associated with frequent immune-related adverse events. Although baseline immune characteristics have been associated with clinical outcomes, little is known about the early immune dynamics induced by this therapy. Here, we investigated whether longitudinal clinical monitoring and high-dimensional profiling of blood circulating T cells could identify early biomarkers of response or toxicity during treatment. Our results indicate that all treated patients exhibit an early depletion of circulating T cells associated with T-cell activation within the first two weeks. Integration of clinical and immunological parameters using Factorial Analysis of Mixed Data (FAMD) identified immune features associated with treatment outcome. Responders had lower plasma soluble BCMA concentrations, fewer bone lesions, higher circulating lymphocyte counts at baseline. During the first days of treatment, responders exhibited a more pronounced increase in plasma CXCL10 levels, associated with a greater decrease in T lymphocyte counts. Overall, our findings suggest that integrating clinical and immune parameters measured during the first days of treatment may enable early patient stratification and support the development of a predictive score to identify patients with multiple myeloma who are most likely to benefit from CD3xBCMA BsAb therapy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/743749v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1cb079org.highwire.dtl.DTLVardef@1860106org.highwire.dtl.DTLVardef@ad36d3org.highwire.dtl.DTLVardef@1ea5c1e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIIntegrated clinical and blood T-cell immune profiling using FAMD enables patient stratification following CD3xBCMA BsAb therapy. C_LIO_LIT-cell immune activation occurs predominantly within the first two weeks of therapy. C_LIO_LIFirst-week clinical and immune parameters identify patients most likely to benefit from therapy. C_LIO_LIHigh CXCL10 levels, a profound early decline in circulating T cells, low sBCMA levels, and fewer bone lesions are candidate predictive markers of treatment response. C_LI

Endothelial-to-mesenchymal transition (EndMT) has become a central mechanism in developmental biology, fibrosis, vascular disease, and cancer. We previously reported on an integrated signaling model in which TGF-{beta}2 induces EndMT through coordinated activation of Smad-dependent and Smad-independent signaling pathways converging on Snail, while GSK-3{beta} regulates Snail activity. We performed a systematic figure-by-figure reproducibility analysis of the original publication. Independent studies published between 2011 and 2026 were identified and curated according to predefined inclusion criteria. Each original experimental conclusion was evaluated for independent confirmation. In parallel, selected biochemical experiments were independently reproduced using newly acquired reagents and contemporary Western blot methodologies. Independent publications consistently reproduced each major mechanistic conclusion of the original study, including activation of Smad, ERK, PI3K/AKT, and p38 MAPK signaling, regulation of Snail expression, EndMT-associated marker switching, and GSK-3{beta}-dependent control of Snail activity. Independent laboratory experiments reproduced the principal biochemical findings using contemporary reagents and experimental workflows. The combined literature analysis and independent laboratory replication demonstrate that the mechanistic framework in our previous study has remained reproducible across multiple laboratories, endothelial cell types, disease models, and fifteen years of investigation. This work illustrates a complementary framework for assessing reproducibility that integrates direct experimental replication with cumulative independent validation.

Colorectal cancer remains among the most lethal malignancies worldwide, and the proliferative programme that sustains it has proved to be a challenging target, particularly with acceptable selectivity. Herein, we combined stage-resolved transcriptomic analysis with experimental testing in colorectal cancer cells to inquire whether small molecules, in particular melatonin, act on that programme. The comparison of stage II, III and IV colorectal tumours with normal tissue identified 410 genes upregulated at every stage as a core set, dominated by cell-cycle, spindle-assembly and chromosome-segregation functions. Twenty hub genes were extracted from the corresponding protein interaction network, thirteen of which were required for viability across 59 colorectal cancer cell lines in genome-wide CRISPR screening data. Target-set enrichment nominated E2F4, FOXM1, SIN3A and both DNA-binding subunits of NF-Y as upstream regulators. NF-YA and NF-YB were distinctive in one respect: their annotated targets include BUB1 and CCNA2 but exclude NCAPG, yielding a testable prediction. Our experimental results showed melatonin reduces SW480 viability with an IC of 2.63 mM and lowers BUB1 and CCNA2 expression in different manners of concentration-dependency, while NCAPG remains unchanged. Melatonin treatment arrests cells in G1 phase, causes a drastic fall in the cycling S-phase fraction, impairs the migration and proliferation phenotype, and rises apoptosis moderately. We also found {beta}2-microglobulin to be an unsuitable normalization reference gene for CRC research due to changes upon treatment. Selective repression of two NF-Y targets with sparing of a non-target is consistent with reduced NF-Y-dependent transcription, though occupancy and subunit-level evidence are to be established.

BackgroundCardiac fibrosis drives adverse myocardial remodelling through persistent fibroblast activation, ECM deposition, and impaired cardiac function. Current therapies offer limited protection against cardiac fibrosis progression. Elafibranor is a dual PPAR-/{delta} agonist approved for the treatment of liver disease. However, its effects in human models of cardiac fibrosis remain insufficiently explored. MethodsElafibranor was evaluated in complementary human in vitro TGF-{beta}1-induced cardiac fibrosis models: 2D primary fibroblasts, 3D fibroblast spheroids, spontaneously contracting 3D cardiac microtissues, and hiPSC-derived cardiomyocytes. Viability, apoptosis, fibroblast activation, ECM remodelling, mitochondrial respiration, nucleotide and NAD pools, calcium handling, contractility, and transcriptomic profiles were assessed. ResultsAt non-cytotoxic concentrations, elafibranor attenuated TGF-{beta}1-driven cardiac fibrosis responses. In 2D cardiac fibroblasts, it reduced myofibroblast differentiation, procollagen 11 secretion, and partially restored mitochondrial respiratory capacity. In 3D spheroids, it preserved viability, attenuated caspase-3/7 activation, and suppressed procollagen 11 release. In cardiac microtissues, elafibranor reduced ECM accumulation, shifted transcriptomic profiles toward redox-metabolic/cytoprotective pathways, altered adenine nucleotide and NAD pools, and partially recovered contraction parameters. In hiPSC-derived cardiomyocytes, elafibranor modulated calcium handling, contractility, and mitochondrial respiration. ConclusionsElafibranor mitigates TGF-{beta}1-driven cardiac fibrosis by suppressing fibroblast activation and ECM remodelling while promoting adaptive metabolic, redox, and bioenergetic responses, supporting balanced PPAR-/{delta} activation as a potential therapeutic strategy for cardiac fibrosis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/745425v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1cbd94eorg.highwire.dtl.DTLVardef@27a44borg.highwire.dtl.DTLVardef@9354baorg.highwire.dtl.DTLVardef@9f9946_HPS_FORMAT_FIGEXP M_FIG C_FIG

Different conformations, or strains, of -synuclein (-syn) aggregates are believed to be responsible for the distinct seeding propensities, propagation profiles, and clinical presentations in Lewy body diseases (LBD) and multiple system atrophy (MSA). While biochemical properties and strain differences of insoluble deposits have been extensively characterized, the understanding of what influence soluble -syn species may have on these processes is limited to a small number of studies focusing on complex mixtures of soluble species or on a single - synucleinopathy. Given that soluble oligomers are considered highly pathologically relevant, we isolated and characterized the biochemical, seeding, and toxicity properties of size-fractionated soluble -syn species from MSA and LBD brains, comparing them to species from control brains without known neurological disease (Ctrl). We observed that levels of differently sized oligomers phosphorylated at Ser129, as well as soluble large oligomers (>450 kDa), were increased in LBD compared to both MSA and Ctrl brains. Nevertheless, species derived from MSA brain exhibited seeding activity across the spectrum of -syn species (oligomers, monomers, and truncated forms) in the seed amplification assay, whereas only oligomeric species (>150 kDa) from LBD cases were seeding-prone. In the HEK293 -syn (A53T)-YFP biosensor line, as well as in murine primary neurons, only large oligomers (>450 kDa) from MSA cases induced seeding and aggregation of -syn. Taken together, our study suggests that soluble -syn species derived from MSA and LBD brains show different biochemical, aggregation and seeding patterns, presumably due to strain variations of the respective oligomers. Our findings provide novel insight into the pathogenesis of different -synucleinopathies, which may guide us in the development of targeted therapeutics.

The methyltransferase EZH2 (Enhancer of Zest Homolog 2) and the demethylase KDM6B (Lysine Demethylase 6B) have been associated with epithelial to mesenchymal transition (EMT) and poor prognosis in various cancers. These enzymes methylate and demethylate H3K27me3 and regulate distinct sets of genes controlling EMT induction, despite having opposite catalytic activities. This could be due to their recruitment or the modulation of their activity by partner proteins on specific loci. This work sought to identify proteins associated with chromatin and interacting with EZH2 or with KDM6B during EMT. To do so, co-immunoprecipitation and mass spectroscopy was used under TGF{beta} (Tumor growth factor {beta}) and TNF (Tumor necrosis factor ) treatment to induce EMT in A549 lung cancer cells. Surprisingly, numerous proteins related to focal adhesions were identified to interact with EZH2 or KDM6B. These proteins are part of a nuclear protein interaction network previously described as nucleo-adhesome. Among these proteins, TGFB1I1 (transforming growth factor induced peptide 1) and CSRP2 (cysteine and glycine rich protein 2) were further confirmed to interact with KDM6B in the nucleus and even more so during EMT. The target genes of these complexes were then sought by knocking down KDM6B, TGFB1I1 or CSRP2. Three genes (coding Integrin alpha 5, Laminin y2 and Matrix Metalloproteinase 9) were confirmed to be regulated by KDM6B, TGFB1I1 and CSRP2. These findings may have clinical relevance, as immunohistochemistry analyses performed on a cohort of lung cancer patients revealed increased nuclear localization of TGFB1I1 and CSRP2 in cells undergoing EMT.

The timing of DNA replication and centrosome duplication is tightly regulated with cell cycle progression to ensure the faithful duplication of the genome during cell division. Both DNA and centrosomes are licensed for replication in late telophase/early G1, replicated in S phase and segregated during mitosis; yet how defects in DNA replication licensing are coupled to centrosome homeostasis remains poorly understood. Here, we show that depletion of the replication licensing inhibitor Geminin in proliferating mouse embryonic fibroblasts induces robust centrosome amplification together with impaired primary cilium assembly. Rather than promoting whole-genome reduplication, knockdown of Geminin triggers a replication stress response, characterized by DNA damage accumulation throughout the cycle, and activation of an ATR-dependent DNA damage response. Mechanistically, Geminin depletion-induced replication stress activates the ATR-Chk1-Wee1 checkpoint axis prolonging G2 and leading to premature centriole disengagement and centrosome amplification. These findings identify replication stress as the signaling module that couples defective DNA replication licensing to centrosome amplification.

BackgroundOwing to their potent immunomodulatory properties, mesenchymal stromal cell (MSC)-derived small extracellular vesicles (EVs) have emerged as promising neuroprotective treatments for ischemic stroke. Preclinical studies using MSC-EVs have mainly been performed in young, otherwise healthy rodents. Stroke patients frequently carry vascular risk factors and comorbidities. We herein investigated whether MSC-EVs retain neuroprotective activity in hyperlipidemic mice on cholesterol-rich Western diet. MethodsMale C57BL/6J mice were exposed to regular normal diet or Western diet for 6 weeks. At the age of 9-10 weeks, mice were exposed to transient intraluminal middle cerebral artery occlusion (MCAO). Vehicle or MSC-EVs (2x106 or 6x106 cell equivalents) were intravenously administered immediately after reperfusion, and vehicle or rosuvastatin (5 mg/kg/day) were intraperitoneally applied starting immediately after or seven days before MCAO. Neurological deficits, ischemic injury, and immune responses were evaluated up to 72 hours post-ischemia. To investigate the hyperlipidemia-associated immune dysregulation, mice received DNase-I before or immediately after MCAO. In defined subgroups, monocytes/ macrophages or neutrophils were additionally depleted by clodronate liposomes or anti-Ly6G antibodies, respectively. ResultsIn contrast to normolipidemic control mice, MSC-EVs failed to induce post-ischemic neuroprotection in hyperlipidemic mice. Neither MSC-EV dose escalation nor rosuvastatin co-treatment restored the therapeutic efficacy of MSC-EVs. Hyperlipidemia induced systemic innate immune dysregulation characterized by reduced monocyte/ macrophage activation, increased neutrophil activation, and elevated circulating cell-free DNA. DNase-I treatment before, but not after MCAO reversed these immune abnormalities and restored neuroprotection by MSC-EVs, decreasing neurological deficits, infarct volume and brain edema. Depletion of either monocytes/ macrophages or neutrophils abolished the neuroprotective effects of MSC-EVs in DNase-I-pretreated hyperlipidemic mice. ConclusionsImmune dysregulation abolishes MSC-EV-induced neuroprotection after ischemic stroke in hyperlipidemic mice. DNase-I priming restores MSC-EV responsiveness through mechanisms critically involving monocyte/ macrophage and neutrophil rebalancing. Our data highlight the host immune status as determinant of EV therapeutic efficacy.

Traumatic brain injury (TBI) impacts millions of individuals annually causing death, disability, and a heightened risk for long-term neurological and neuropsychiatric disorders. In recent years the fruit fly, Drosophila melanogaster has become a valuable model organism to study the cellular and molecular responses following TBI. AP-1 mediated transcriptional responses to TBI have previously been identified in Drosophila using pan-glial approaches. Fruit flies possess multiple glial subtypes which vary greatly in both cellular morphology and function, including glia of the blood hemolymph barrier, cortex, astrocyte-like, and ensheathing glia. By generating and utilizing a nuclear localized AP-1 transcriptional reporter, we identified glial subtype-specific differences in the extent of AP-1 activation following injury. Our findings identify a strong AP-1 response in the blood hemolymph barrier and ensheathing glia, a moderate response in cortex glia and little to no AP-1 activation in astrocyte-like glia. In addition, we inhibited AP-1 signaling in each glial subtype and tested the effect on acute survival. We found that inhibition of the AP-1 response in neuropil ensheathing glia leads to increased mortality following mild and moderate TBI. These results show that AP-1 activation levels vary across glial subtypes after TBI, with activation in neuropil ensheathing glia having a particularly important role in promoting post-injury survival. ARTICLE SUMMARYUsing Drosophila as a model organism, we investigated the early molecular and cellular response to traumatic brain injury. Our findings substantiate the requirement of a functional glial associated AP-1 transcriptional activation response for survival. Using colocalization studies, we characterized the AP-1 glial response in six morphologically and functionally distinct glia subtypes. After TBI, we find high levels of AP-1 activation in glia of the hemolymph brain barrier, cortex glia, and ensheathing glia. We further show the importance of AP-1 transcription within the neuropil ensheathing glia subtype for optimal survival following TBI.

Mutations in p53 and its 12 isoforms can alter its functions. As N-terminally truncated isoforms of p53 (delta40p53, delta133p53, and delta160p53) participate in tetramer formation, they are important regulators of cancer fate. Although delta40p53- and delta133p53-mediated regulation of cancer is well reported, the mechanism underlying delta160p53 production and its functional role remains unclear. We investigated the internal ribosomal entry site (IRES)-mediated translation of {Delta}160p53 and its role in cancer regulation. As differential synthesis of delta160p53 was observed under different stress conditions, IRES-mediated translation of this isoform was demonstrated using bicistronic luciferase constructs. No cryptic promoters or splicing sites were detected in the IRES sequence. Cell death and late apoptosis were significantly decreased, while proliferation, the number of cells in the S phase, and drug resistance were induced by delta160p53. Furthermore, delta160p53 did not induce p53-responsive promoters. RNA sequencing analysis of delta160p53 overexpression showed similar results, along with the inhibition of other tumor suppressor genes. Overall, our results provide insights into IRES-mediated translation of delta160p53, which can be considered a novel target for cancer treatment.

Regucalcin (RGN) plays diverse roles in cell biology, highlighting its importance in both physiological and pathological conditions. Prostate cancer patients with higher RGN expression exhibited significantly longer disease-free survival. Although RGN is a cell signaling suppressor, the molecular mechanisms underlying tumor suppression by RGN in the tumor microenvironment through cell-cell communication remain unclear. PC3 prostate cancer cell lines stably expressing RGN or a control vector were generated for this study. Extracellular vesicles (EVs) were isolated from these cell lines using differential ultracentrifugation. The murine macrophage cell line J7441 was treated with isolated EVs, and effects on M2 polarization were evaluated using qRT-PCR and western blot analysis. To assess the potential anti-tumor effects of EVs, PC3 parental cells were subcutaneously implanted at two sites per mouse, followed by intratumoral injection of the respective EVs. Tumor volume was monitored. Harvested fresh frozen tumor tissues underwent immunofluorescence staining for CD206, an M2 macrophage marker. RGN was detected in EVs from RGN-expressing cells, and treatment with these RGN-containing EVs was associated with reduced tumor growth and reduced M2 macrophage polarization in vitro and in vivo. Furthermore, recombinant RGN protein reduced the levels of p-AKT1 and p-ERK1/2. Moreover, the suppression of M2 macrophage polarization by RGN-containing EVs was accompanied by decreased p-AKT1 and p-ERK1/2 in vitro. This study describes an EV-associated mechanism that may contribute to the regulation of macrophage polarization and indicates that RGN-containing EVs merit further evaluation as a candidate approach for cancer treatment. Causal validation, such as macrophage depletion or CD206 knockdown, and evaluation in additional models remain to be addressed in future studies.

At the onset of intervertebral disc degeneration, within the disc core, the pH and osmolarity decrease, and the residing notochordal cells (NCs) gradually transition towards nucleus pulposus cells (NPCs). How these microenvironmental cues shape the NCs extracellular vesicles (EV)-enriched secretome, and thus EV-mediated communication with NPCs during this transition, remains poorly understood. To study this, we collected the secretome from pig NC-rich tissue cultured for 4 days in either healthy or degenerate disc media to mimic these changes. In both conditions, NC-rich tissues were largely comparable at the histological and biochemical levels. Despite, tissues released glycosaminoglycans (GAGs), depleting the extracellular matrix. Surprisingly, degenerative media did not differentially release inflammatory regulators, though it reduced PGE2 release. We asked whether this extended to EV-enriched secretome media (SM_EV+), and found that the degenerative media reduced the number of EVs without altering their morphology or size. We then determined NC-EV association of inflammatory and matrix regulators. NC-EV isolation enriched MMP1, IL6 and IL10 and depleted soluble GAGs. Conversely, EV-depletion (SM_EV-) removed most GAGs without affecting MMP1, IL6, and IL10, suggesting that they contribute to the NC-EV soft corona. Functionally, healthy SM_EV+ improved GAG production by NPCs, but attenuated TBXT expression. Degenerate SM_EV+ did not elicit detectable EV-specific effects. These findings suggest that, in health, secretome-mediated communication from NCs to NPCs is only partially EV-mediated. At the onset of IVD degeneration, low pH and osmolarity impair the release of NC-EVs and negate the EV-specific beneficial matrix-anabolic effects on NPCs, contributing to the NC-to-NPC transition.

Urothelial cancer (UC) cells of the luminal subtype exhibit partial, incomplete differentiation towards umbrella cells that line bladder lumen, seen by morphology and gene expression. Differentiation is stalled even though the cells express master transcription factors (MTFs) that drive luminal urothelial differentiation, e.g., FOXA1 and CEBPB, at levels seen in normal differentiated urothelium. We therefore analyzed the FOXA1/CEBPB MTF hub by mass spectrometry. SWI/SNF coactivator complex (CoA) components, e.g., SMARCA4, ARID1A, that read the epigenetic activation mark histone 3 lysine 27 acetylation (H3K27ac) and use ATP-hydrolysis to open chromatin, were the most abundant proteins pulled-down with FOXA1/CEBPB. However, genes for these and other CoA, e.g., CREBBP, EP300 that write H3K27ac, were mutated/deleted in >95% of UCs in clinical series. Also contained in the hub were corepressors (CoR) that erase H3K27ac and close chromatin, e.g., HDAC1, CHD4 - genes for these CoR were recurrently gained in UCs. Chromatin analyses showed H3K27ac-centered remodeling was needed to activate umbrella but not constitutively accessible cell growth/division/housekeeping genes. Restoring ARID1A into ARID1A-mutated UC cells using lentiviral transduction, or inhibiting CoR with siRNA or small molecules, activated umbrella genes and terminated replications. In summary, UC-genesis selects for loss- and gain-of-function of CoA and CoR respectively in the urothelial-lineage MTF hub; small molecule CoR-inhibitors are candidate remedies to renew maturation towards terminal differentiated-fates. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/744501v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@19e853org.highwire.dtl.DTLVardef@e3c934org.highwire.dtl.DTLVardef@ae7874org.highwire.dtl.DTLVardef@66331b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background and AimsMutations in the desmin (DES) gene cause a variety of cardiomyopathies associated with arrhythmias, yet the electrophysiological consequences of these variants remain largely uncharacterized. The aim of this study was to investigate the pathogenic mechanisms of the de novo DES p.R406W variant, which was identified in a 9-year-old patient who suffered from severe ventricular arrhythmias and sudden cardiac death without overt structural heart disease. MethodsHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the DES p.R406W variant (including the patients line) were compared to isogenic controls. Action potentials (AP) of hiPSC-CMs were recorded using patch-clamp. Furthermore, 3D engineered heart tissues (EHTs) were generated from hiPSC-CMs and their APs were recorded with sharp microelectrodes. Analytical techniques also included transmission electron microscopy (TEM) and integrated transcriptomic and proteomic profiling. Finally, a heterozygous knock-in (KI) mouse model carrying the Des p.R405W ortholog was evaluated through surface ECG, echocardiography and ex vivo cardiac optical mapping. ResultsThe DES p.R406W mutation prolonged AP duration in IM-R406W hiPSC-CMs and EHTs vs Control ones. Multi-omics analysis of EHTs revealed a dysregulation of genes and proteins involved in contractile function, cell adhesion, and electrical activity. TEM imaging revealed changes in Z-disc architecture in mutant tissues. Twenty-week-old Des p.R405W KI mice exhibited ventricular conduction slowing (prolonged QRS) and a high susceptibility to ventricular tachyarrhythmias, likely due to reentrant mechanisms. Mild hypertrophy was also observed, but only in females. ConclusionThe DES p.R406W variant is highly pathogenic, causing electrical and structural remodeling of the myocardium. This study highlights the effectiveness of hiPSC-CMs and EHTs in recapitulating the clinical phenotype of desminopathy, providing a platform for investigating the mechanisms of early-onset cardiac arrhythmias and SCD.