The transcriptome of early compensatory kidney growth reveals cell and time specific responses.

Following kidney removal, the remaining kidney enlarges and increases its function. The mechanism and signals driving this compensatory kidney hypertrophy and the enlargement of its constituent kidney cells remains elusive. RNA-seq studies in mice undergoing hypertrophy 24, 48, and 72 h following nephrectomy were undertaken to understand the early transcriptional changes. This revealed substantial enhancement of cholesterol biosynthesis pathways, increases in mitochondrial gene expression and cell cycle perturbations. Single nuclei RNA-seq delineated cell specific changes at 24 h post nephrectomy and showed that sterol binding protein 2 (SREBP2) activity increases in medullary thick ascending limb cells in keeping with promotion of cholesterol synthesis. Cultured renal tubular cells were examined for insulin-like growth factor-1 (IGF-1) stimulated hypertrophy and SREBP2 was found to be required for increase in cell size. This work describes the early cell specific growth pathways mediating cellular and kidney hypertrophy with an intriguing role for cholesterol synthesis.

Intergenerational epigenetic inheritance mediated by MYS-2/MOF in the pathogenesis of Alzheimer's disease.

Although autosomal-dominant inheritance is believed an important cause of familial clustering Alzheimer's disease (FAD), it covers only a small proportion of FAD incidence, and so we investigated epigenetic memory as an alternative mechanism to contribute for intergenerational AD pathogenesis. Our data in vivo showed that mys-2 of Caenorhabditis elegans that encodes a putative MYST acetyltransferase responsible for H4K16 acetylation modulated AD occurrence. The phenotypic improvements in the parent generation caused by mys-2 disfunction were passed to their progeny due to epigenetic memory, which resulted in similar H4K16ac levels among the candidate target genes of MYS-2 and similar gene expression patterns of the AD-related pathways. Furthermore, the ROS/CDK-5/ATM pathway functioned as an upstream activator of MYS-2. Our study indicated that MYS-2/MOF could be inherited intergenerationally via epigenetic mechanisms in C. elegans and mammalian cell of AD model, providing a new insight into our understanding of the etiology and inheritance of FAD.

Proteomic landscape of tunneling nanotubes reveals CD9 and CD81 tetraspanins as key regulators.

Tunneling nanotubes (TNTs) are open actin- and membrane-based channels, connecting remote cells and allowing direct transfer of cellular material (e.g. vesicles, mRNAs, protein aggregates) from the cytoplasm to the cytoplasm. Although they are important especially, in pathological conditions (e.g. cancers, neurodegenerative diseases), their precise composition and their regulation were still poorly described. Here, using a biochemical approach allowing to separate TNTs from cell bodies and from extracellular vesicles and particles (EVPs), we obtained the full composition of TNTs compared to EVPs. We then focused on two major components of our proteomic data, the CD9 and CD81 tetraspanins, and further investigated their specific roles in TNT formation and function. We show that these two tetraspanins have distinct non-redundant functions: CD9 participates in stabilizing TNTs, whereas CD81 expression is required to allow the functional transfer of vesicles in the newly formed TNTs, possibly by regulating docking to or fusion with the opposing cell.

MMP9 drives ferroptosis by regulating GPX4 and iron signaling.

Ferroptosis, defined by the suppression of glutathione peroxidase-4 (GPX4) and iron overload, is a distinctive form of regulated cell death. Our in-depth research identifies matrix metalloproteinase-9 (MMP9) as a critical modulator of ferroptosis through its influence on GPX4 and iron homeostasis. Employing an innovative MMP9 construct without collagenase activity, we reveal that active MMP9 interacts with GPX4 and glutathione reductase, reducing GPX4 expression and activity. Furthermore, MMP9 suppresses key transcription factors (SP1, CREB1, NRF2, FOXO3, and ATF4), alongside GPX1 and ferroptosis suppressor protein-1 (FSP1), thereby disrupting the cellular redox balance. MMP9 regulates iron metabolism by modulating iron import, storage, and export via a network of protein interactions. LC-MS/MS has identified 83 proteins that interact with MMP9 at subcellular levels, implicating them in ferroptosis regulation. Integrated pathway analysis (IPA) highlights MMP9's extensive influence on ferroptosis pathways, underscoring its potential as a therapeutic target in conditions with altered redox homeostasis and iron metabolism.

Legionella pneumophila modulates macrophage functions through epigenetic reprogramming via the C-type lectin receptor Mincle.

Legionella pneumophila is a pathogen which can lead to a severe form of pneumonia in humans known as Legionnaires disease after replication in alveolar macrophages. Viable L. pneumophila actively secrete effector molecules to modulate the host's immune response. Here, we report that L. pneumophila-derived factors reprogram macrophages into a tolerogenic state, a process to which the C-type lectin receptor Mincle (CLEC4E) markedly contributes. The underlying epigenetic state is characterized by increases of the closing mark H3K9me3 and decreases of the opening mark H3K4me3, subsequently leading to the reduced secretion of the cytokines TNF, IL-6, IL-12, the production of reactive oxygen species, and cell-surface expression of MHC-II and CD80 upon re-stimulation. In summary, these findings provide important implications for our understanding of Legionellosis and the contribution of Mincle to reprogramming of macrophages by L. pneumophila.

Epitranscriptomic reader YTHDF2 regulates SEK1(MAP2K4)-JNK-cJUN inflammatory signaling in astrocytes during neurotoxic stress.

As the most abundant glial cells in the central nervous system (CNS), astrocytes dynamically respond to neurotoxic stress, however, the key molecular regulators controlling the inflammatory status of these sentinels during neurotoxic stress are many and complex. Herein, we demonstrate that the m6A epitranscriptomic mRNA modification tightly regulates the pro-inflammatory functions of astrocytes. Specifically, the astrocytic neurotoxic stressor, manganese (Mn), downregulated the m6A reader YTHDF2 in human and mouse astrocyte cultures and in the mouse brain. Functionally, YTHDF2 knockdown augmented, while its overexpression dampened, the neurotoxic stress-induced proinflammatory response, suggesting YTHDF2 serves as a key upstream regulator of inflammatory responses in astrocytes. Mechanistically, YTHDF2 RIP-sequencing identified MAP2K4 (MKK4; SEK1) mRNA as a YTHDF2 target influencing inflammatory signaling. Our target validation revealed that Mn-exposed astrocytes mediate proinflammatory responses by activating the phosphorylation of SEK1, JNK, and cJUN signaling. Collectively, YTHDF2 serves as a key upstream 'molecular switch' controlling SEK1(MAP2K4)-JNK-cJUN proinflammatory signaling in astrocytes.

GRP94 promotes anoikis resistance and peritoneal metastasis through YAP/TEAD1 pathway in gastric cancer.

Anoikis resistance allows cancer cells to avoid death caused by detachment from the extracellular matrix's adhesion, enabling these cells to infiltrate and migrate to regions such as the peritoneum. This study emphasizes GRP94's involvement in anoikis resistance and peritoneal metastasis in gastric cancer (GC). It's found that GRP94 overexpression, linked to poor prognosis, was potentially due to SP1 and GRP94 promoter interactions, confirmed through dual luciferase reporter (DLR), chromatin immunoprecipitation (ChIP), and quantitative real-time PCR (real-time qPCR). Increased GRP94 enhanced GC cells' anoikis resistance and metastasis. Decreasing GRP94 had opposite effects, potentially through yes-associated protein (YAP)/TEAD1 axis inhibition, with raised YAP phosphorylation and decreased TEAD1 levels detected by western blotting (WB). Inhibiting YAP counteracted GRP94's effects on anoikis resistance and metastasis, while activating YAP reversed the effects of GRP94 reduction. Animal experiments verified GRP94's contribution to GC's peritoneal metastasis. In conclusion, our work highlights the effect of GRP94 on anoikis resistance, showing potential value in treating peritoneal metastasis of GC.

Patient-derived tumor organoids mimic treatment-induced DNA damage response in glioblastoma.

Glioblastoma (GB) is the most common primary malignant brain tumor, characterized by resistance to therapy. Despite aggressive treatment options, GB remains an incurable disease. Invasiveness and heterogeneity are key GB features that cannot be studied in preclinical in vitro models. In this study, we investigated the effects of standard therapy using patient-derived GB organoids (GBOs). GBOs reflect the complexity and heterogeneity of the original tumor tissue. No significant effect on GBO viability or invasion was observed after irradiation and temozolomide treatment. E3 ubiquitin-protein ligase (MDM2), cyclin-dependent kinase inhibitor 1A (CDKN1A), and the serine/threonine kinases ATM and ATR were upregulated at the gene and protein levels after treatment. Our results show that the p53 pathway and DNA-damage response mechanisms were triggered, suggesting that GBOs recapitulate GB therapy resistance. GBOs thus provide a highly efficient platform to assess the specific responses of GB patients to therapy and to further explore therapy resistance.

Defective CFTR modulates mechanosensitive channels TRPV4 and PIEZO1 and drives endothelial barrier failure.

Cystic fibrosis (CF) is a genetic disease caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Despite reports of CFTR expression on endothelial cells, pulmonary vascular perturbations, and perfusion deficits in CF patients, the mechanism of pulmonary vascular disease in CF remains unclear. Here, our pilot study of 40 CF patients reveals a loss of small pulmonary blood vessels in patients with severe lung disease. Using a vessel-on-a-chip model, we establish a shear-stress-dependent mechanism of endothelial barrier failure in CF involving TRPV4, a mechanosensitive channel. Furthermore, we demonstrate that CFTR deficiency downregulates the function of PIEZO1, another mechanosensitive channel involved in angiogenesis and wound repair, and exacerbates loss of small pulmonary blood vessel. We also show that CFTR directly interacts with PIEZO1 and enhances its function. Our study identifies key cellular targets to mitigate loss of small pulmonary blood vessels in CF.

Paternal hypercholesterolemia elicits sex-specific exacerbation of atherosclerosis in offspring.

Emerging studies suggest that various parental exposures affect offspring cardiovascular health, yet the specific mechanisms, particularly the influence of paternal cardiovascular disease (CVD) risk factors on offspring cardiovascular health, remain elusive. The present study explores how paternal hypercholesterolemia affects offspring atherosclerosis development using the LDL receptor-deficient (LDLR-/-) mouse model. We found that paternal high-cholesterol diet feeding led to significantly increased atherosclerosis in F1 female, but not male, LDLR-/- offspring. Transcriptomic analysis highlighted that paternal hypercholesterolemia stimulated proatherogenic genes, including Ccn1 and Ccn2, in the intima of female offspring. Sperm small noncoding RNAs (sncRNAs), particularly transfer RNA-derived (tRNA-derived) small RNAs (tsRNAs) and rRNA-derived small RNAs (rsRNAs), contribute to the intergenerational transmission of paternally acquired metabolic phenotypes. Using a newly developed PANDORA-Seq method, we identified that high-cholesterol feeding elicited changes in sperm tsRNA/rsRNA profiles that were undetectable by traditional RNA-Seq, and these altered sperm sncRNAs were potentially key factors mediating paternal hypercholesterolemia-elicited atherogenesis in offspring. Interestingly, high-cholesterol feeding altered sncRNA biogenesis-related gene expression in the epididymis but not testis of LDLR-/- sires; this may have led to the modified sperm sncRNA landscape. Our results underscore the sex-specific intergenerational effect of paternal hypercholesterolemia on offspring cardiovascular health and contribute to the understanding of chronic disease etiology originating from parental exposures.

S-acylation of NLRP3 provides a nigericin sensitive gating mechanism that controls access to the Golgi.

NLRP3 is an inflammasome seeding pattern recognition receptor activated in response to multiple danger signals which perturb intracellular homeostasis. Electrostatic interactions between the NLRP3 polybasic (PB) region and negatively charged lipids on the trans-Golgi network (TGN) have been proposed to recruit NLRP3 to the TGN. In this study, we demonstrate that membrane association of NLRP3 is critically dependant on S-acylation of a highly conserved cysteine residue (Cys-130), which traps NLRP3 in a dynamic S-acylation cycle at the Golgi, and a series of hydrophobic residues preceding Cys-130 which act in conjunction with the PB region to facilitate Cys-130 dependent Golgi enrichment. Due to segregation from Golgi localised thioesterase enzymes caused by a nigericin induced breakdown in Golgi organisation and function, NLRP3 becomes immobilised on the Golgi through reduced de-acylation of its Cys-130 lipid anchor, suggesting that disruptions in Golgi homeostasis are conveyed to NLRP3 through its acylation state. Thus, our work defines a nigericin sensitive S-acylation cycle that gates access of NLRP3 to the Golgi.

Upregulated expression of ubiquitin ligase TRIM21 promotes PKM2 nuclear translocation and astrocyte activation in experimental autoimmune encephalomyelitis.

Reactive astrocytes play critical roles in the occurrence of various neurological diseases such as multiple sclerosis. Activation of astrocytes is often accompanied by a glycolysis-dominant metabolic switch. However, the role and molecular mechanism of metabolic reprogramming in activation of astrocytes have not been clarified. Here, we found that PKM2, a rate-limiting enzyme of glycolysis, displayed nuclear translocation in astrocytes of EAE (experimental autoimmune encephalomyelitis) mice, an animal model of multiple sclerosis. Prevention of PKM2 nuclear import by DASA-58 significantly reduced the activation of mice primary astrocytes, which was observed by decreased proliferation, glycolysis and secretion of inflammatory cytokines. Most importantly, we identified the ubiquitination-mediated regulation of PKM2 nuclear import by ubiquitin ligase TRIM21. TRIM21 interacted with PKM2, promoted its nuclear translocation and stimulated its nuclear activity to phosphorylate STAT3, NF-κB and interact with c-myc. Further single-cell RNA sequencing and immunofluorescence staining demonstrated that TRIM21 expression was upregulated in astrocytes of EAE. TRIM21 overexpressing in mice primary astrocytes enhanced PKM2-dependent glycolysis and proliferation, which could be reversed by DASA-58. Moreover, intracerebroventricular injection of a lentiviral vector to knockdown TRIM21 in astrocytes or intraperitoneal injection of TEPP-46, which inhibit the nuclear translocation of PKM2, effectively decreased disease severity, CNS inflammation and demyelination in EAE. Collectively, our study provides novel insights into the pathological function of nuclear glycolytic enzyme PKM2 and ubiquitination-mediated regulatory mechanism that are involved in astrocyte activation. Targeting this axis may be a potential therapeutic strategy for the treatment of astrocyte-involved neurological disease.

Stearic acid alleviates aortic medial degeneration through maintaining mitochondrial dynamics homeostasis via inhibiting JNK/MAPK signaling.

Aortic dissection is characterized pathologically by aortic medial degeneration (AMD) where disturbance of mitochondrial dynamics may be involved. Stearic acid (SA) can promote mitochondrial fusion and improve mitochondrial function. Here, we established an AMD mouse model through oral administration of ß-aminopropionitrile (BAPN) and a cellular model by treating primary vascular smooth muscle cells (VSMCs) with Angiotensin-II to explore the potential role of SA in AMD. Our results showed SA reduced AMD and prolonged survival of BAPN-treated mice. Excessive mitochondrial fission was observed during AMD both in vivo and in vitro, and SA reduced mitochondrial fission and increased fusion. Additionally, SA promoted expression of contractile phenotype markers of VSMCs. At the molecular level, SA reduced AMD by inhibiting JNK/MAPK signaling. Our study suggests SA can promote mitochondrial fusion and increase the contractile phenotype of VSMCs by inhibiting JNK/MAPK signaling, thereby reducing AMD formation and possibly the consequent risk of aortic dissection.

Multi-omics analysis of aggregative multicellularity.

All organisms have to carefully regulate their gene expression, not least during development. mRNA levels are often used as proxy for protein output; however, this approach ignores post-transcriptional effects. In particular, mRNA-protein correlation remains elusive for organisms that exhibit aggregative rather than clonal multicellularity. We addressed this issue by generating a paired transcriptomics and proteomics time series during the transition from uni-to multicellular stage in the social ameba Dictyostelium discoideum. Our data reveals that mRNA and protein levels correlate highly during unicellular growth, but decrease when multicellular development is initiated. This accentuates that transcripts alone cannot accurately predict protein levels. The dataset provides a useful resource to study gene expression during aggregative multicellular development. Additionally, our study provides an example of how to analyze and visualize mRNA and protein levels, which should be broadly applicable to other organisms and conditions.

Rosiglitazone retards the progression of iron overload-induced osteoarthritis by impeding chondrocyte ferroptosis.

Ferroptosis is implicated in several diseases, including iron overload-induced osteoarthritis (IOOA), which is marked by oxidative stress, iron imbalance, and lipid peroxidation. Given rosiglitazone's (RSG) ability to inhibit lipid peroxidation and ferroptosis, this study aims to assess its therapeutic potential for treating IOOA. Our in vitro results show that RSG targets acyl-CoA synthetase long-chain family member 4 to mitigate impairments induced by interleukin-1 beta and ferric ammonium citrate, including cell apoptosis, senescence, inflammatory responses, extracellular matrix degradation, and ferroptosis. RSG reduced intracellular iron content, alleviated oxidative stress and lipid peroxidation, mitigated damage to membrane-bound organelles, and enhanced glucose transport. Additionally, pre-treatment with RSG imparted anti-ferroptotic properties to chondrocytes. In vivo, RSG alleviated cartilage degradation, inflammatory responses, and ferroptosis in mice with IOOA. In conclusion, RSG exhibits chondroprotective and anti-ferroptotic effects by suppressing lipid peroxidation and restoring iron homeostasis, highlighting its potential for treating IOOA.