Alexander disease (AxD) is a rare and severe neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP). While the exact disease mechanism remains unknown, previous studies suggest that mutant GFAP influences many cellular processes, including cytoskeleton stability, mechanosensing, metabolism, and proteasome function. While most studies have primarily focused on GFAP-expressing astrocytes, GFAP is also expressed by radial glia and neural progenitor cells, prompting questions about the impact of GFAP mutations on central nervous system (CNS) development. In this study, we observed impaired differentiation of astrocytes and neurons in co-cultures of astrocytes and neurons, as well as in neural organoids, both generated from AxD patient-derived induced pluripotent stem (iPS) cells with a GFAPR239C mutation. Leveraging single-cell RNA sequencing (scRNA-seq), we identified distinct cell populations and transcriptomic differences between the mutant GFAP cultures and a corrected isogenic control. These findings were supported by results obtained with immunocytochemistry and proteomics. In co-cultures, the GFAPR239C mutation resulted in an increased abundance of immature cells, while in unguided neural organoids and cortical organoids, we observed altered lineage commitment and reduced abundance of astrocytes. Gene expression analysis revealed increased stress susceptibility, cytoskeletal abnormalities, and altered extracellular matrix and cell-cell communication patterns in the AxD cultures, which also exhibited higher cell death after stress. Overall, our results point to altered cell differentiation in AxD patient-derived iPS-cell models, opening new avenues for AxD research.
- MeSH
- Alexander Disease * genetics pathology metabolism MeSH
- Astrocytes * metabolism pathology MeSH
- Cell Differentiation * physiology MeSH
- Glial Fibrillary Acidic Protein * metabolism genetics MeSH
- Induced Pluripotent Stem Cells * metabolism MeSH
- Coculture Techniques MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Mutation MeSH
- Neural Stem Cells metabolism MeSH
- Neurons metabolism pathology MeSH
- Organoids metabolism pathology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: Natural killer (NK) cell-based therapies represent a promising approach for acute myeloid leukemia (AML) relapse, yet their efficacy is hindered by immunosuppressive factors such as transforming growth factor beta (TGF-β) in the tumor microenvironment. This study investigated the effects of TGF-β on NK cell cytotoxicity and migration using 2D and 3D co-culture models that mimic the leukemic microenvironment. METHODS: TGF-β production was evaluated in AML-derived leukemic cell lines and mesenchymal stromal cells (hTERT-MSCs) using ELISA. Bulk RNA sequencing (RNA-seq) was performed to analyze global gene expression changes in TGF-β-treated primary human NK cells. NK cell cytotoxicity and migration were assessed in 2D monolayer and 3D spheroid co-cultures containing hTERT-MSCs and leukemic cells using flow cytometry and confocal microscopy. RESULTS: Both leukemic cells and MSCs produced TGF-β, with increased levels observed in MSCs after co-culture with primary AML blasts. RNA sequencing revealed that TGF-β altered key gene pathways associated with NK cell cytotoxicity, adhesion, and migration, supporting its immunosuppressive role. In functional assays, TGF-β exposure significantly reduced NK cell-mediated cytotoxicity in a time-dependent manner and impaired NK cell infiltration into 3D spheroids, particularly in models incorporating MSCs. Additionally, MSCs themselves provided a protective environment for leukemic cells, further reducing NK cell effectiveness in 2D co-cultures. CONCLUSION: TGF-β suppresses both NK cell cytotoxicity and migration, limiting their ability to eliminate leukemic cells and infiltrate the bone marrow niche (BMN). These findings provide novel insights into TGF-β-mediated immune evasion mechanisms and provide important insights for the future design of NK-based immunotherapies and clinical trials.
- Publication type
- Journal Article MeSH
BACKGROUND: The progression and recurrence are the fatal prognostic factors in glioma patients. However, the therapeutic role and potential mechanism of TRAF7 in glioma patients remain largely unknown. METHODS: TRAF7 RNA-seq was analysed with the TCGA and CGGA databases between glioma tissues and normal brain tissues. The expression of TRAF7, cellular senescence and cell cycle arrest pathways in glioma tissues and cell lines was detected by real-time quantitative PCR (RT-qPCR), western blotting and immunohistochemistry. The interaction between TRAF7 and KLF4 was determined by Co-immunoprecipitation (Co-IP) assays. The functions of TRAF7 combined with lomustine in glioma were assessed by both in vitro, in vivo and patient-derived primary and recurrent glioma stem cell (GSC) assays. RESULTS: High TRAF7 expression is closely associated with a higher recurrence rate and poorer overall survival (OS). In vitro, TRAF7 knockdown significantly inhibits glioma cell proliferation, invasion, and migration. RNA-seq analysis revealed that TRAF7 inhibition activates pathways related to cellular senescence and cell cycle arrest. In both in vitro and patient-derived GSC assays, the combination of sh-TRAF7 and lomustine enhanced therapeutic efficacy by inducing senescence and G0/G1 cell cycle arrest, surpassing the effects of lomustine or TRAF7 inhibition alone. Mechanistically, TRAF7 interacts with KLF4, and a rescue assay demonstrated that KLF4 overexpression could reverse the effects of TRAF7 depletion on proliferation and cellular senescence. In vivo, TRAF7 knockdown combined with lomustine treatment effectively suppressed glioma growth. CONCLUSION: TRAF7 could be used as a predictive biomarker and the potential therapeutic target among National Comprehensive Cancer Network (NCCN) treatment guidelines in the progression and recurrence of glioma. Lomustine, regulating cellular senescence and cell cycle could be the priority choice in glioma patients with high-level TRAF7 expression.
- MeSH
- Gene Knockdown Techniques MeSH
- Glioma * pathology genetics drug therapy metabolism MeSH
- Kruppel-Like Factor 4 MeSH
- Humans MeSH
- Neoplasm Recurrence, Local * genetics pathology MeSH
- Lomustine * pharmacology therapeutic use MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Brain Neoplasms * pathology genetics drug therapy metabolism MeSH
- Tumor Necrosis Factor Receptor-Associated Peptides and Proteins * genetics metabolism MeSH
- Prognosis MeSH
- Disease Progression MeSH
- Cell Proliferation MeSH
- Gene Expression Regulation, Neoplastic MeSH
- Cellular Senescence * drug effects MeSH
- Xenograft Model Antitumor Assays MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: Early detection of colorectal cancer (CRC) significantly improves its management and patients' survival. Circular RNAs (circRNAs) are peculiar covalently closed transcripts involved in gene expression modulation whose dysregulation has been extensively reported in CRC cells. However, little is known about their alterations in the early phases of colorectal carcinogenesis. METHODS: In this study, we performed an integrative analysis of circRNA profiles in RNA-sequencing (RNA-Seq) data of 96 colorectal cancers, 27 adenomas, and matched adjacent mucosa tissues. We also investigated the levels of cognate linear transcripts and those of regulating RNA-binding proteins (RBPs). Levels of circRNA-interacting microRNAs (miRNAs) were explored by integrating data of small RNA-Seq performed on the same samples. RESULTS: Our results revealed a significant dysregulation of 34 circRNAs (paired adj. p < 0.05), almost exclusively downregulated in tumor tissues and, prevalently, in early disease stages. This downregulation was associated with decreased expression of circRNA host genes and those encoding for RBPs involved in circRNA biogenesis, including NOVA1, RBMS3, and MBNL1. Guilt-by-association analysis showed that dysregulated circRNAs correlated with increased predicted activity of cell proliferation, DNA repair, and c-Myc signaling pathways. Functional analysis showed interactions among dysregulated circRNAs, RBPs, and miRNAs, which were supported by significant correlations among their expression levels. Findings were validated in independent cohorts and public datasets, and the downregulation of circLPAR1(2,3) and circLINC00632(5) was validated by ddPCR. CONCLUSIONS: These results support that multiple altered regulatory mechanisms may contribute to the reduction of circRNA levels that characterize early colorectal carcinogenesis.
- Publication type
- Journal Article MeSH
Single-cell RNA-seq methods can be used to delineate cell types and states at unprecedented resolution but do little to explain why certain genes are expressed. Single-cell ATAC-seq and multiome (ATAC + RNA) have emerged to give a complementary view of the cell state. It is however unclear what additional information can be extracted from ATAC-seq data besides transcription factor binding sites. Here, we show that ATAC-seq telomere-like reads counter-inituively cannot be used to infer telomere length, as they mostly originate from the subtelomere, but can be used as a biomarker for chromatin condensation. Using long-read sequencing, we further show that modern hyperactive Tn5 does not duplicate 9 bp of its target sequence, contrary to common belief. We provide a new tool, Telomemore, which can quantify nonaligning subtelomeric reads. By analyzing several public datasets and generating new multiome fibroblast and B-cell atlases, we show how this new readout can aid single-cell data interpretation. We show how drivers of condensation processes can be inferred, and how it complements common RNA-seq-based cell cycle inference, which fails for monocytes. Telomemore-based analysis of the condensation state is thus a valuable complement to the single-cell analysis toolbox.
- MeSH
- Single-Cell Analysis * methods MeSH
- B-Lymphocytes metabolism cytology MeSH
- Cell Cycle * genetics MeSH
- Chromatin Immunoprecipitation Sequencing methods MeSH
- Chromatin * metabolism chemistry genetics MeSH
- Fibroblasts metabolism cytology MeSH
- Humans MeSH
- RNA-Seq methods MeSH
- Telomere * genetics MeSH
- Binding Sites MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
INTRODUCTION: Stem cells derived from adipose tissue are gaining popularity in the field of regenerative medicine due to their adaptability and clinical potential. Their rapid growth, ability to differentiate, and easy extraction with minimal complications make adipose-derived stem cells (ADSCs) a promising option for many treatments, particularly those targeting bone-related diseases. This study analyzed gene expression in canine ADSCs subjected to long-term culture and osteogenic differentiation. METHODS: ADSCs were isolated from discarded surgical waste and cultured for 14 days with and without differentiation media to assess osteogenic changes. RNA sequencing (RNA-seq) and bioinformatical analysis were performed to obtain comprehensive transcriptomic data. A total of 17793 genes were detected and GO enrichment analysis was performed on the differentially expressed genes to identify significantly up- and downregulated Biological Process (BP) GO terms across each comparison. RESULTS: The upregulation of apoptosis-regulating genes and genes related to circulatory system development suggest an induction of these processes, while the downregulation of neurogenesis and gliogenesis genes points to reciprocal regulation during osteogenic differentiation of canine ADSCs. DISCUSSION: These findings underscore the potential of ADSCs in bone regeneration and offer valuable insights for advancing tissue engineering, however further studies, including proteomic analyses, are needed to confirm these patterns and their biological significance.
- Publication type
- Journal Article MeSH
The lateral line system enables fishes and aquatic-stage amphibians to detect local water movement via mechanosensory hair cells in neuromasts, and many species to detect weak electric fields via electroreceptors (modified hair cells) in ampullary organs. Both neuromasts and ampullary organs develop from lateral line placodes, but the molecular mechanisms underpinning ampullary organ formation are understudied relative to neuromasts. This is because the ancestral lineages of zebrafish (teleosts) and Xenopus (frogs) independently lost electroreception. We identified Bmp5 as a promising candidate via differential RNA-seq in an electroreceptive ray-finned fish, the Mississippi paddlefish (Polyodon spathula; Modrell et al., 2017, eLife 6: e24197). In an experimentally tractable relative, the sterlet sturgeon (Acipenser ruthenus), we found that Bmp5 and four other Bmp pathway genes are expressed in the developing lateral line, and that Bmp signalling is active. Furthermore, CRISPR/Cas9-mediated mutagenesis targeting Bmp5 in G0-injected sterlet embryos resulted in fewer ampullary organs. Conversely, when Bmp signalling was inhibited by DMH1 treatment shortly before the formation of ampullary organ primordia, supernumerary ampullary organs developed. These data suggest that Bmp5 promotes ampullary organ development, whereas Bmp signalling via another ligand(s) prevents their overproduction. Taken together, this demonstrates opposing roles for Bmp signalling during ampullary organ formation.
- MeSH
- Bone Morphogenetic Proteins * metabolism genetics MeSH
- Lateral Line System * embryology metabolism MeSH
- Fish Proteins metabolism genetics MeSH
- Fishes genetics MeSH
- Signal Transduction * MeSH
- Gene Expression Regulation, Developmental MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
The adaptive immune response critically hinges on the functionality of T cell receptors, governed by complex molecular mechanisms, including ubiquitination. In this study, we delved into the role of in T cell immunity, focusing on T cell-B cell conjugate formation and T cell activation. Using a CRISPR-Cas9 screening approach targeting deubiquitinases genes in Jurkat T cells, we identified BAP1 as a key positive regulator of T cell-B cell conjugate formation. Subsequent investigations into BAP1 knockout cells revealed impaired T cell activation, evidenced by decreased MAPK and NF-kB signaling pathways and reduced CD69 expression upon T cell receptor stimulation. Flow cytometry and qPCR analyses demonstrated that BAP1 deficiency leads to decreased surface expression of T cell receptor complex components and reduced mRNA levels of the co-stimulatory molecule CD28. Notably, the observed phenotypes associated with BAP1 knockout are specific to T cells and fully dependent on BAP1 catalytic activity. In-depth RNA-seq and mass spectrometry analyses further revealed that BAP1 deficiency induces broad mRNA and protein expression changes. Overall, our findings elucidate the vital role of BAP1 in T cell biology, especially in T cell-B cell conjugate formation and T cell activation, offering new insights and directions for future research in immune regulation.
- MeSH
- Lymphocyte Activation * immunology MeSH
- B-Lymphocytes * immunology metabolism MeSH
- Jurkat Cells MeSH
- Humans MeSH
- Tumor Suppressor Proteins * metabolism genetics MeSH
- Receptors, Antigen, T-Cell * metabolism MeSH
- Signal Transduction MeSH
- T-Lymphocytes * immunology metabolism MeSH
- Ubiquitin Thiolesterase * genetics metabolism deficiency MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Familial dysautonomia is a debilitating congenital neurodegenerative disorder with no causative therapy. It is caused by a homozygous mutation in ELP1 gene, resulting in the production of the transcript lacking exon 20. The compounds studied as potential treatments include the clinical candidate kinetin, a plant hormone from the cytokinin family. We explored the relationship between the structure of a set of kinetin derivatives (N = 72) and their ability to correct aberrant splicing of the ELP1 gene. Active compounds can be obtained by the substitution of the purine ring with chlorine and fluorine at the C2 atom, with a small alkyl group at the N7 atom, or with diverse groups at the C8 atom. On the other hand, a substitution at the N3 or N9 atoms resulted in a loss of activity. We successfully tested a hypothesis inspired by the remarkable tolerance of the position C8 to substitution, postulating that the imidazole of the purine moiety is not required for the activity. We also evaluated the activity of phytohormones from other families, but none of them corrected ELP1 mRNA aberrant splicing. A panel of in vitro ADME assays, including evaluation of transport across model barriers, stability in plasma and in the presence of liver microsomal fraction as well as plasma protein binding, was used for an initial estimation of the potential bioavailability of the active compounds. Finally, a RNA-seq data suggest that 8-aminokinetin modulates expression spliceosome components.
- MeSH
- Kinetin * pharmacology chemistry MeSH
- Humans MeSH
- Molecular Structure MeSH
- RNA Precursors * genetics metabolism MeSH
- RNA Splicing * drug effects MeSH
- Transcriptional Elongation Factors metabolism genetics MeSH
- Dose-Response Relationship, Drug MeSH
- Structure-Activity Relationship MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Adenosine deaminase acting on RNA 1 (ADAR1) is the principal enzyme for the adenosine-to-inosine RNA editing that prevents the aberrant activation of cytosolic nucleic acid sensors by endogenous double stranded RNAs and the activation of interferon-stimulated genes. In mice, the conditional neural crest deletion of Adar1 reduces the survival of melanocytes and alters the differentiation of Schwann cells that fail to myelinate nerve fibers in the peripheral nervous system. These myelination defects are partially rescued upon the concomitant removal of the Mda5 antiviral dsRNA sensor in vitro, suggesting implication of the Mda5/Mavs pathway and downstream effectors in the genesis of Adar1 mutant phenotypes. By analyzing RNA-Seq data from the sciatic nerves of mouse pups after conditional neural crest deletion of Adar1 (Adar1cKO), we here identified the transcription factors deregulated in Adar1cKO mutants compared to the controls. Through Adar1;Mavs and Adar1cKO;Egr1 double-mutant mouse rescue analyses, we then highlighted that the aberrant activation of the Mavs adapter protein and overexpression of the early growth response 1 (EGR1) transcription factor contribute to the Adar1 deletion associated defects in Schwann cell development in vivo. In silico and in vitro gene regulation studies additionally suggested that EGR1 might mediate this inhibitory effect through the aberrant regulation of EGR2-regulated myelin genes. We thus demonstrate the role of the Mda5/Mavs pathway, but also that of the Schwann cell transcription factors in Adar1-associated peripheral myelination defects.
- MeSH
- Adenosine Deaminase * genetics metabolism MeSH
- Cell Differentiation * genetics MeSH
- Neural Crest * metabolism MeSH
- Interferon-Induced Helicase, IFIH1 genetics metabolism MeSH
- Myelin Sheath metabolism MeSH
- Mice, Knockout * MeSH
- Mice MeSH
- Schwann Cells * metabolism pathology MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
