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The chromosome 15q11.2 locus is deleted in 1.5% of patients with genetic epilepsy and confers a risk for intellectual disability and schizophrenia. Individuals with this deletion demonstrate increased cortical thickness, decreased cortical surface area and white matter abnormalities. Human induced pluripotent stem cell (iPSC)-derived neural progenitor cells from 15q11.2 deletion individuals exhibit early adhesion junction and migration abnormalities, but later neuronal development and function have not been fully assessed. Imaging studies indicating altered structure and network connectivity in the anterior brain regions and the cingulum suggest that in addition to alterations in neural progenitor dynamics, there may also be structural and functional changes within discrete networks of neurons. To explore this, we generated human forebrain cortical neurons from iPSCs derived from individuals with or without 15q11.2 deletion and used longitudinal imaging and multielectrode array analysis to evaluate neuronal development over time. 15q11.2 deleted neurons exhibited fewer connections and an increase in inhibitory neurons. Individual neurons had decreased neurite complexity and overall decreased neurite length. These structural changes were associated with a reduction in multiunit action potential generation, bursting and synchronization. The 15q11.2 deleted neurons also demonstrated specific functional deficits in glutamate- and GABA-mediated neuronal network activity and synchronization with a delay in the maturation of the inhibitory response to GABA. These data indicate that deletion of the 15q11.2 region is sufficient to impair the structural and functional maturation of cortical neuron networks, and suggest an in vitro correlate to the pathologic changes in humans with the 15q11.2 deletion.

The accumulation of foamy macrophages is a pathological hallmark of demyelinating brain disorders. Perturbed metabolism and efflux of intracellular lipids underlie the development of a harmful foamy macrophage phenotype in these disorders, yet, the molecular mechanisms underlying this dysregulation are poorly understood. Here, we show that the ubiquitin-proteasome system controls the turnover of the cholesterol efflux transporter ATP-binding cassette A1 (ABCA1) in lipid-loaded macrophages in the brain. We report that accumulation of myelin-derived lipids promotes the abundance and activity of ubiquitin-protein E3 ligase A (UBE3A) in macrophages, which stimulates ABCA1 ubiquitination and subsequent degradation. This boosts cellular lipid accumulation and induces an inflammatory macrophage phenotype that impairs remyelination. We further establish Tat-interacting protein 30 (TIP30), an inhibitor of importin β-mediated nuclear import, as an essential regulator of cytosolic UBE3A levels. Together, our findings identify UBE3A as a driver of foam cell formation and indicate that targeting UBE3A-mediated ABCA1 degradation is a promising strategy to enhance central nervous system repair. Foamy macrophages are a pathological hallmark of demyelinating brain disorders. Here, the authors identify the molecular mechanisms underlying the faulty regulation of lipid efflux that cause accumulation, suggesting a promising strategy to enhance central nervous system repair.

The mechanisms through which mutations in splicing factor genes drive clonal hematopoiesis (CH) and myeloid malignancies, and their close association with advanced age, remain poorly understood. Here we show that telomere maintenance plays an important role in this phenomenon. First, by studying 454,098 UK Biobank participants, we find that, unlike most CH subtypes, splicing-factor-mutant CH is more common in those with shorter genetically predicted telomeres, as is CH with mutations in PPM1D and the TERT gene promoter. We go on to show that telomere attrition becomes an instrument for clonal selection in advanced age, with splicing factor mutations ‘rescuing’ HSCs from critical telomere shortening. Our findings expose the lifelong influence of telomere maintenance on hematopoiesis and identify a potential shared mechanism through which different splicing factor mutations drive leukemogenesis. Understanding the mechanistic basis of these observations can open new therapeutic avenues against splicing-factor-mutant CH and hematological or other cancers. This study explores the relationship between telomere length and clonal hematopoiesis. Splicing factor and PPM1D gene mutations are more frequent in people with genetically predicted shorter telomere lengths, suggesting that these mutations protect against the consequences of telomere attrition.

Caveolin-1 (CAV1), a pivotal protein implicated in endothelial cell-mediated angiogenesis, assumes an ambiguous role with elusive underlying mechanisms in the pathogenesis of inflammatory bowel disease (IBD). In this investigation, we delineated the involvement of CAV1 in murine models of dextran sulfate sodium (DSS)-induced colitis. CAV1 knockout mice manifested attenuated pathological and inflammatory damage to the epithelium, whereas mice overexpressing CAV1 exhibited contrasting outcomes. In vivo, the accumulation of epithelial CAV1 contributed to the disruption of the epithelial barrier by promoting necroptosis. Subsequent mechanistic analyses revealed that the colitis-protective protein DJ-1 regulated CAV1 through a proteasome-mediated protein degradation pathway. Utilizing necroptosis-modeled organoids from murine intestines and pharmacological inhibition of necroptosis, our findings demonstrated that the DJ-1/CAV1 pathway governed epithelial inflammation via necroptosis in the context of colitis. In summary, our research revealed that epithelial CAV1 aggravated necroptosis in experimental colitis, leading to impairment of the epithelial barrier, which was negatively regulated by DJ-1.

The placenta is a dynamic, embryo-derived organ essential for fetal growth and development. While all eutherian mammals have placentas composed of fetal-derived trophoblasts that mediate maternal–fetal exchange, their anatomical and histological structures vary across species due to evolutionary divergence. Despite the cellular heterogeneity of porcine trophoblasts in vivo, understanding the mechanisms driving porcine placental development has been limited by the lack of in vitro models replicating this heterogeneity. In this study, we derived swine trophoblast organoids (sTOs) from full-term porcine placentas, retaining key transcriptional signatures of in vivo trophoblasts. To identify conserved cell populations, we integrated Visium spatial transcriptomics from mid-gestation porcine placentas with single-cell transcriptomics from sTOs. Spatial transcriptomics revealed novel markers of the porcine uterus and placenta, enabling precise separation of histological structures at the maternal–fetal interface. The integration of tissue and sTO transcriptomics showed that sTOs spontaneously differentiate into distinct trophoblast populations, with conserved gene expression and cell communication programs. These findings demonstrate that sTOs recapitulate porcine placental trophoblast populations, offering a powerful model for advancing placentation research. Our work also provides a spatially resolved whole-transcriptome dataset of the porcine maternal–fetal interface, opening new avenues for discoveries in placental development, evolution, and health across mammals. Understanding porcine placental development has been hindered by the lack of in vitro models that reflect its cellular heterogeneity. This study develops a swine trophoblast organoid model that mimics in vivo trophoblast diversity and gene expression, providing a novel platform for studying placental biology and maternal-fetal interactions.

Regulatory T cells (Tregs) are essential for maintaining immune balance by controlling the activation and expansion of other immune cells. Conventional suppression assays often rely on co-culturing purified cell populations, which limits multiplexed phenotyping and physiological relevance. This protocol describes a high-dimensional, single-cell assay for profiling Treg-mediated suppression within a peripheral blood mononuclear cell (PBMC) system. Tregs are first isolated by cell sorting and then reintroduced into autologous PBMCs at defined ratios. A 52-marker mass cytometry (CyTOF) panel is used to quantify cell division and phenotypic responses across multiple immune subsets. This approach allows for integrated analysis of Treg function with broad compatibility for patient profiling and drug evaluation. Key features • Quantifies Treg-mediated suppression in autologous PBMCs at single-cell resolution.• Enables high-dimensional phenotyping and proliferation tracking across multiple immune subsets using a 52-marker CyTOF panel.• Maintains physiological relevance by assessing suppression in a complex PBMC environment.• Compatible with patient-derived samples and drug perturbation experiments for translational immunology applications

Simple SummaryPU.1 is a critical transcription factor involved in the development and function of macrophages that play a central role in tumor initiation and progression. However, its role in tumor-associated macrophages (TAMs) remains poorly understood. This study investigates the impact of PU.1 inhibition using the chemical inhibitor DB2313 in a mouse tumor model. The findings aim to offer a potential therapeutic strategy for targeting PU.1 in TAMs to suppress tumor growth. AbstractBackground: Targeting tumor-associated macrophages (TAMs) is a promising immunotherapy for cancers, but current strategies are limited due to strategic caveats. PU.1 is a transcription factor required for macrophage generation and differentiation. To date, the effect of PU.1 inhibition on solid tumors is unknown. Methods: This study examines the anti-tumor effect of PU.1 inhibition and its mechanism using the small-molecule DB2313 in mouse melanoma and breast tumor models. Results: We found that inhibition of PU.1 by DB2313 suppresses B16-OVA melanoma and 4T1 breast tumor growth in mice. In the melanoma tumor model, DB2313 enhanced tumor recruitment of CD4+ T helper 1 (Th1) and cytotoxic T/natural killer (NK) cells by targeting TAMs. Transcriptome and targeted gene expression analyses revealed that PU.1 inhibition by DB2313 and small-interference RNAs enhances CXCL9 expression in bulk tumors, TAMs, and bone marrow-derived macrophages. The anti-tumor effects of DB2313 were abolished by depleting macrophages with clodronate or inhibiting the CXCL9-CXCR3 chemokine axis using CXCL9- or CXCR3-neutralizing antibodies. Conclusions: These results suggest that pharmacological inhibition of PU.1 suppresses tumor growth by at least promoting the infiltration of lymphocytes into tumors through the CXCL9-CXCR3 chemokine axis. Our study establishes a framework for developing TAM-modulating immunotherapies by targeting the transcriptional factor PU.1.

Over 95% of ependymomas that arise in the cortex are driven by a gene fusion involving the zinc finger translocation-associated (ZFTA) protein. Here, using super-resolution and lattice light-sheet microscopy, we demonstrate that the most frequent fusion variant, ZFTA–RELA (ZR), forms dynamic nuclear condensates that are required for oncogene expression and tumorigenesis. Mutagenesis studies of ZR reveal a key intrinsically disordered region (IDR) in RELA that governs condensate formation. Condensate-modulating IDR mutations introduced into ZR impaired its genomic occupancy at oncogenic loci and inhibited the recruitment of transcriptional effector proteins, such as MED1, BRD4 and RNA polymerase II. Using nuclear magnetic resonance spectroscopy, we examined the DNA-binding residues of the critical zinc finger (ZF1) found in ZR and characterized their significance for condensate formation, genomic binding and oncogene activation. We generated synthetic ZFTA fusion proteins where IDRs from known condensate-forming proteins were grafted into ZR. Synthetic ZFTA fusion oncoproteins utilizing IDRs from EWS and FUS restored condensate formation, oncogene transcription and tumour initiation in mice. These findings provide key insights into the oncogenic mechanism of ZR and the importance of IDR acquisition in fusion oncoproteins in brain cancer. Arabzade, Shirnekhi, Varadharajan, Ippagunta and colleagues show that the zinc finger translocation-associated (ZFTA) fusion oncoproteins gain intrinsically disordered regions, inducing nuclear condensate formation and oncogenic activation.

The majority of immune-mediated disease (IMD) risk loci are located in non-coding regions of the genome, making it difficult to decipher their functional effects in relevant physiological contexts. To assess the extent to which alternative splicing contributes to IMD risk, we mapped genetic variants associated with alternative splicing (splicing quantitative trait loci or sQTL) in macrophages exposed to a wide range of environmental stimuli. We found that genes involved in innate immune response pathways undergo extensive differential splicing in response to stimulation and detected significant sQTL effects for over 5734 genes across all stimulation conditions. We colocalised sQTL signals for over 700 genes with IMD-associated risk loci from 22 IMDs with high confidence (PP4 ≥ 0.75). Approximately half of the colocalisations implicate lowly-used splice junctions (mean usage ratio <0.1). Finally, we demonstrate how an inflammatory bowel disease (IBD) risk allele increases the usage of a lowly-used isoform of PTPN2, a negative regulator of inflammation. Together, our findings highlight the role alternative splicing plays in IMD risk, and suggest that lowly-used splicing events significantly contribute to complex disease risk. The authors show that alternative splicing is an important layer of macrophage response to environmental stimuli. Genetic determinants of this response, often targeting low-usage splicing events, are linked to several immune-mediated diseases.

Recent evidence has highlighted immune checkpoint inhibitors as among the most promising immunotherapies for various malignancies. However, a significant proportion of HCC patients exhibit poor responses. Lipid metabolic heterogeneity is considered a key driver of cancer progression. However, the role of lipid metabolic reprogramming in HCC immunotherapy resistance remains poorly understood. Herein, we aimed to illuminate the potential relationship between lipid metabolic reprogramming and ICI resistance and provide novel strategies to increase the HCC immunotherapy response. Patients who received PD-1/PD-L1 inhibitors were enrolled. The effect of TACC3 on the tumor microenvironment was validated via single-cell RNA sequencing in HCC-bearing mouse models. Targeted metabolomics was performed to analyze the regulatory role of TACC3 in HCC metabolism. To address HCC immunotherapy resistance, we developed a targeted nucleic acid therapeutic utilizing N-acetylgalactosamine (GalNAc) to conjugate siTACC3. Through clinical cohort analysis, we found that TACC3 was overexpressed in HCC patients with poor response to immunotherapy. Furthermore, we demonstrated that silencing tumor-derived TACC3 optimizes the cytotoxicity of infiltrating CD8+ T lymphocytes. Both in vitro and in vivo assays suggested that TACC3 maintains ACSL4-mediated polyunsaturated fatty acid (PUFA) metabolism in HCC cells. Additionally, TACC3 accelerates ACSL4 expression by interacting with LARP1 and PABPC1, which stabilize ACSL4 mRNA. The results of preclinical models demonstrated the satisfactory efficacy of GalNAc-conjugated siTACC3 combined with PD-1 inhibitor therapy for HCC. In summary, tumor-derived TACC3 impairs the tumor-killing activity of CD8+ T lymphocytes through PUFA metabolism-associated crosstalk. Targeting TACC3 represents a novel and practicable strategy to augment ICI efficacy against HCC.

The immune response against pathogens involves multiple cell state transitions and complex gene expression changes. Here, we establish a single-cell in vivo new RNA labeling sequencing method (scIVNL-seq) and apply it to survey time-resolved RNA dynamics during immune response to acute enteric infection with Salmonella. We show that the detection of new RNA synthesis reflects more realistic information on cell activation and gene transcription than total RNA level. Interplay of RNA synthesis and degradation modulates the dynamics of total RNA. The bone marrow macrophages are first primed at a very early stage upon Salmonella infection. In contrast, the innate immune response of macrophages in intestine is limited. Notably, intestinal CD8+ T cells and plasma cells are rapidly and specifically activated at the early stage post infection. Intestinal late enterocytes quickly express MHC-I molecules and present Salmonella antigen to CD8+ T cells for their activation, serving as antigen presenting cells for the initiation of adaptive immunity. Our findings reveal the RNA control strategies and the dynamic activation rules of immune cells in response to Salmonella infection, challenging the doctrine boundary between innate immunity and adaptive immunity against bacterial infection. The immune response involves multiple cell state transitions and complex gene expression changes. Here, the authors establish scIVNL-seq to survey time-resolved RNA dynamics of immune cells during Salmonella infection, revealing distinct RNA control strategies and cell activation patterns underlying immune responses.

The human nephron is a highly patterned tubular structure that develops specialized cells to regulate bodily fluid homeostasis, blood pressure, and urine secretion throughout life. Approximately 1 million nephrons form in each kidney during embryonic and fetal development, but how they develop is poorly understood. Here, we interrogate axial patterning mechanisms in the human nephron using an iPSC-derived kidney organoid system that generates hundreds of developmentally synchronized nephrons, and we compare it to in vivo human kidney development using single cell and spatial transcriptomic approaches. We show that human nephron patterning is controlled by integrated WNT/BMP/FGF signaling. Imposing a WNTON/BMPOFF state established a distal nephron identity that matures into thick ascending loop of Henle cells by endogenously activating FGF. Simultaneous suppression of FGF signaling switches cells back to a proximal cell-state, a transformation that is in itself dependent on BMP signal transduction. Our system highlights plasticity in axial nephron patterning, delineates the roles of WNT, FGF, and BMP mediated mechanisms controlling nephron patterning, and paves the way for generating nephron cells on demand. A versatile, human iPSC-derived nephron engineering platform that permits scrutiny of axial patterning mechanisms is critical for identifying the origins of human kidney disease. Here they describe a system in which synchronized human nephron structures are generated from pluripotent stem cells, enabling manipulation of axial segmentation.