Cell-specific Pathways Research Articles

Cystic fibrosis-related diabetes is associated with reduced islet protein expression of GLP-1 receptor and perturbation of cell-specific transcriptional programs

Insulin secretion is impaired in individuals with cystic fibrosis (CF), contributing to high rates of CF-related diabetes (CFRD) and substantially increasing disease burden. To develop improved therapies for CFRD, better knowledge of pancreatic pathology in CF is needed. Glucagon like peptide-1 (GLP-1) from islet α cells potentiates insulin secretion by binding GLP-1 receptors (GLP-1Rs) on β cells. We determined whether expression of GLP-1 and/or its signaling components are reduced in CFRD, thereby contributing to impaired insulin secretion. Immunohistochemistry of pancreas from humans with CFRD versus no-CF/no-diabetes revealed no difference in GLP-1 immunoreactivity per islet area, whereas GLP-1R immunoreactivity per islet area or per insulin-positive islet area was reduced in CFRD. Using spatial transcriptomics, we observed several differentially expressed α- and/or β-cell genes between CFRD and control pancreas. In CFRD, we found upregulation of α-cell PCSK1 which encodes the enzyme (PC1/3) that generates GLP-1, and downregulation of α-cell PCSK1N which inhibits PC1/3. Gene set enrichment analysis also revealed α and β cell-specific pathway dysregulation in CFRD. Together, our data suggest intra-islet GLP-1 is not limiting in CFRD, but its action may be restricted due to reduced GLP-1R protein levels. Thus, restoring β-cell GLP-1R protein expression may improve β-cell function in CFRD.

Downregulation of PIK3IP1/TrIP on T cells is controlled by TCR signal strength, PKC and metalloprotease-mediated cleavage.

The protein known as PI3K-interacting protein (PIK3IP1), or transmembrane inhibitor of PI3K (TrIP), is highly expressed by T cells and can modulate PI3K activity in these cells. Several studies have also revealed that TrIP is rapidly downregulated following T cell activation. However, it is unclear as to how this downregulation is controlled. Using a novel monoclonal antibody that robustly stains cell-surface TrIP, we demonstrate that TrIP is lost from the surface of activated T cells in a manner dependent on the strength of signaling through the T cell receptor (TCR) and specific downstream signaling pathways, in particular classical PKC isoforms. TrIP expression returns by 24 hours after stimulation, suggesting that it may play a role in resetting TCR signaling at later time points. We also provide evidence that ADAM family proteases are required for both constitutive and stimulation-induced downregulation of TrIP in T cells. Finally, by expressing truncated forms of TrIP in cells, we identify the region in the extracellular stalk domain of TrIP that is targeted for proteolytic cleavage.

Downregulation of PIK3IP1/TrIP on T cells is controlled by TCR signal strength, PKC and metalloprotease-mediated cleavage

The protein known as PI3K-interacting protein (PIK3IP1), or transmembrane inhibitor of PI3K (TrIP), is highly expressed by T cells and can modulate PI3K activity in these cells. Several studies have also revealed that TrIP is rapidly downregulated following T cell activation. However, it is unclear as to how this downregulation is controlled. Using a novel monoclonal antibody that robustly stains cell-surface TrIP, we demonstrate that TrIP is lost from the surface of activated T cells in a manner dependent on the strength of signaling through the T cell receptor (TCR) and specific downstream signaling pathways, in particular classical PKC isoforms. TrIP expression returns by 24 hours after stimulation, suggesting that it may play a role in resetting TCR signaling at later time points. We also provide evidence that ADAM family proteases are required for both constitutive and stimulation-induced downregulation of TrIP in T cells. Finally, by expressing truncated forms of TrIP in cells, we identify the region in the extracellular stalk domain of TrIP that is targeted for proteolytic cleavage.

Natural killer cell effector function is critical for host defense against alcohol-associated bacterial pneumonia

Alcohol use is an independent risk factor for the development of bacterial pneumonia due, in part, to impaired mucus-facilitated clearance, macrophage phagocytosis, and recruitment of neutrophils. Alcohol consumption is also known to reduce peripheral natural killer (NK) cell numbers and compromise NK cell cytolytic activity, especially NK cells with a mature phenotype. However, the role of innate lymphocytes, such as NK cells during host defense against alcohol-associated bacterial pneumonia is essentially unknown. We have previously shown that indole supplementation mitigates increases in pulmonary bacterial burden and improves pulmonary NK cell recruitment in alcohol-fed mice, which were dependent on aryl hydrocarbon receptor (AhR) signaling. Employing a binge-on-chronic alcohol-feeding model we sought to define the role and interaction of indole and NK cells during pulmonary host defense against alcohol-associated pneumonia. We demonstrate that alcohol dysregulates NK cell effector function and pulmonary recruitment via alterations in two key signaling pathways. We found that alcohol increases transforming growth factor beta (TGF-β) signaling while suppressing AhR signaling. We further demonstrated that NK cells isolated from alcohol-fed mice have a reduced ability to kill Klebsiella pneumoniae. NK cell migratory capacity to chemokines was also significantly altered by alcohol, as NK cells isolated from alcohol-fed mice exhibited preferential migration in response to CXCR3 chemokines but exhibited reduced migration in response to CCR2, CXCR4, and CX3CR1 chemokines. Together this data suggests that alcohol disrupts NK cell-specific TGF-β and AhR signaling pathways leading to decreased pulmonary recruitment and cytolytic activity thereby increasing susceptibility to alcohol-associated bacterial pneumonia.

Transcriptional landscape of a hypoxia response identifies cell-specific pathways for adaptation.

How the HIF-1 (Hypoxia-Inducible) transcription factor drives and coordinates distinct responses to low oxygen across diverse cell types is poorly understood. We present a multi-tissue single-cell gene-expression atlas of the hypoxia response of the nematode Caenorhabditis elegans . This atlas highlights how cell-type-specific HIF-1 responses overlap and diverge among and within neuronal, intestinal, and muscle tissues. Using the atlas to guide functional analyses of candidate muscle-specific HIF-1 effectors, we discovered that HIF-1 activation drives downregulation of the tspo-1 ( TSPO, Translocator Protein) gene in vulval muscle cells to modulate a hypoxia-driven change in locomotion caused by contraction of body-wall muscle cells. We further showed that in human cardiomyocytes HIF-1 activation decreases levels of TSPO and thereby alters intracellular cholesterol transport and the mitochondrial network. We suggest that TSPO-1 is an evolutionarily conserved mediator of HIF-1-dependent modulation of muscle and conclude that our gene-expression atlas can help reveal how HIF-1 drives cell-specific adaptations to hypoxia.

Integrated Metabolomics and Transcriptomics Analysis of Anacardic Acid Inhibition of Breast Cancer Cell Viability.

Anacardic acid (AnAc) inhibits the growth of estrogen receptor α (ERα)-positive MCF-7 breast cancer (BC) cells and MDA-MB-231 triple-negative BC (TNBC) cells, without affecting primary breast epithelial cells. RNA sequencing (seq) and network analysis of AnAc-treated MCF-7 and MDA-MB-231 cells suggested that AnAc inhibited lipid biosynthesis and increased endoplasmic reticulum stress. To investigate the impact of AnAc on cellular metabolism, a comprehensive untargeted metabolomics analysis was performed in five independent replicates of control versus AnAc-treated MCF-7 and MDA-MB-231 cells and additional TNBC cell lines: MDA-MB-468, BT-20, and HCC1806. An analysis of the global metabolome identified key metabolic differences between control and AnAc-treated within each BC cell line and between MCF-7 and the TNBC cell lines as well as metabolic diversity among the four TNBC cell lines, reflecting TNBC heterogeneity. AnAc-regulated metabolites were involved in alanine, aspartate, glutamate, and glutathione metabolism; the pentose phosphate pathway; and the citric acid cycle. Integration of the transcriptome and metabolome data for MCF-7 and MDA-MB-231 identified Signal transduction: mTORC1 downstream signaling in both cell lines and additional cell-specific pathways. Together, these data suggest that AnAc treatment differentially alters multiple pools of cellular building blocks, nutrients, and transcripts resulting in reduced BC cell viability.

#162 Single-nucleus RNA-seq of biopsy-confirmed IgA nephropathy reveals cell-specific transcriptomic alteration

Abstract Background and Aims Single-nucleus RNA-seq (SnRNA-seq) is a valuable method for identifying cell-specific transcriptomic alterations in kidney cells. Our objective was to investigate SnRNA-seq data from biopsy-confirmed IgA nephropathy (IgAN) cases and identify cell-specific alterations in the transcriptome. Method We collected snap-frozen kidney biopsy tissues from 6 cases of IgA nephropathy (IgAN) and 7 nephrectomy control cases. The disease control group included 3 cases of diabetic kidney disease, 6 cases of minimal change disease, and 6 PLA2R-Ab positive membranous nephropathy cases. SnRNA-seq was performed on the kidney tissues, and kidney cells were identified in the dataset through UMAP clustering. We investigated differences in the proportion of kidney cell types and identified differentially expressed genes (DEGs). Additionally, we searched for representative gene loci previously reported in a multiethnic genome-wide association study (GWAS). Results We successfully collected transcriptomic profiles of >50 000 cells from IgAN, with a considerable enrichment of intraglomerular cells. The DEG analysis revealed a high number of DEGs in mesangial cells, fibroblasts, and vascular smooth muscle cells in the IgAN transcriptome compared to the nephrectomy and disease control cases. In the gene set enrichment analysis, the epithelial-mesenchymal transition pathway was enriched in the glomerular cells of IgAN. Furthermore, among various GWAS loci, ETS1 was the gene significantly highly expressed in the mesangial cells of IgAN, consistent across the compared control groups. Conclusion This investigation represents the largest SnRNA-Seq profiling of IgAN kidney cells to date. We identified notable enriched cell-specific pathways in IgAN. ETS1 may be a kidney-indwelling causal factor for IgAN pathophysiology, considering its notable significance in both previous GWAS results and the current SnRNA-Seq data.

Characterization of Endothelial to Mesenchymal Transition in Air Pollution Mediated Atherosclerosis: Insights from Trajectory Inference and Functional Analyses in Single cell RNA Sequencing Data

Background: Air pollution is the world’s leading environmental risk factor for atherosclerotic cardiovascular disease (ASCVD). Regressive transcriptional reprogramming and transition of cells to primordial states has been identified as a driver of atherosclerosis. Endothelial cell to mesenchymal cell transition (EndoMT) whereby an endothelial cell transforms to a mesenchymal-like cellular phenotype is one such mechanism. However its relevance to air pollution exposure remains uncharacterized. Methods: Male ApoE−/− mice were inhalationally exposed to concentrated particulate matter <2.5μ component (PM2.5) or filtered air (FA, controls) using a Versatile Aerosol Concentration Enrichment System (VACES, 5–6 h/day, 5 d/week for 36 weeks, n = 7/group. PM2.5 concentrations were 70-80 μg/m3 or ~10X ambient levels. Following euthanasia, single-cell RNA-sequencing of aortic cells was performed using the 10-X platform (CA). For all subsequent analyses, aortic cell samples were integrated. Unsupervised clustering (k-NN) and low-dimensional space visualization (UMAP) were performed with a supervised graph method (Minimum Spanning Tree and Principal Curves) to infer and visualize trajectory analysis of novel cell lineages (branching and cell ordering in pseudotime/pseudospace). Functional analyses were carried out by statistical enrichment analysis using Over Representation Analysis (ORA) and Gene Set Enrichment Analysis (GSEA) against databases of Gene Ontologies (GO), Biological Pathways, putative regulatory motifs, proteins, or disease annotations to find over-representation of terms and functions. Pseudotime distribution assessed along exposure groups and cell-types lineages were statistically compared using GLM models. Differential Expression Analysis within lineages and between groups were tested by GLM and GAM models and FDR correction, followed by functional analyses. Results: Comparison of the pseudotime lineage with lineage root node at the endothelial cells revealed a similarity between observed endothelial (EC) to vascular smooth muscle (VSMC) cell transition with the differential transition noted with PM2.5 from FA. This overlap suggests a potential exaggeration of EC to VSMC transition in response to PM2.5 exposure. Differential gene expression profiling upon PM2.5-exposure in EC revealed significant changes in GO terms related to cell differentiation, cell migration, and regulation of cell population proliferation. Specific genes with variable patterns of expression (4481) were identified along the EC to VSMC lineage. KEGG pathway analysis along the lineage revealed enriched pathways including PI3K/Akt signaling pathway, MAPK signaling pathway, and Rap1 signaling pathway which are all implicated in the development and progression of atherosclerosis. Conclusion: Single-cell analysis reveals that chronic exposure to PM2.5 results in the exaggeration of EC to VSMC transition consistent with the EndoMT paradigm that may help explain the acceleration of ASCVD in response to air pollution exposure. Additional analysis of cell specific pathways may provide improved understanding of mechanisms progression of atherosclerosis in response to environmental exposures. NIH: 1R35ES031702-01, R01 HL155450, 3R35ES031702-02S1; Burroughs Wellcome Fund: 1060485. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Multiomic profiling of human clonal hematopoiesis reveals genotype and cell-specific inflammatory pathway activation

AbstractClonal hematopoiesis (CH) is an age-associated phenomenon that increases the risk of hematologic malignancy and cardiovascular disease. CH is thought to enhance disease risk through inflammation in the peripheral blood.1 Here, we profile peripheral blood gene expression in 66 968 single cells from a cohort of 17 patients with CH and 7 controls. Using a novel mitochondrial DNA barcoding approach, we were able to identify and separately compare mutant Tet methylcytosine dioxygenase 2 (TET2) and DNA methyltransferase 3A (DNMT3A) cells with nonmutant counterparts. We discovered the vast majority of mutated cells were in the myeloid compartment. Additionally, patients harboring DNMT3A and TET2 CH mutations possessed a proinflammatory profile in CD14+ monocytes through previously unrecognized pathways such as galectin and macrophage inhibitory factor. We also found that T cells from patients with CH, although mostly unmutated, had decreased expression of GTPase of the immunity associated protein genes, which are critical to T-cell development, suggesting that CH impairs T-cell function.

Identification and functional activity of Nik related kinase (NRK) in benign hyperplastic prostate

ObjectiveBenign prostatic hyperplasia (BPH) is common in elder men. The current study aims to identify differentially expressed genes (DEGs) in hyperplastic prostate and to explore the role of Nik related kinase (NRK) in BPH.MethodsFour datasets including three bulk and one single cell RNA-seq (scRNA-seq) were obtained to perform integrated bioinformatics. Cell clusters and specific metabolism pathways were analyzed. The localization, expression and functional activity of NRK was investigated via RT-PCR, western-blot, immunohistochemical staining, flow cytometry, wound healing assay, transwell assay and CCK-8 assay.ResultsA total of 17 DEGs were identified by merging three bulk RNA-seq datasets. The findings of integrated single-cell analysis showed that NRK remarkably upregulated in fibroblasts and SM cells of hyperplasia prostate. Meanwhile, NRK was upregulated in BPH samples and localized almost in stroma. The expression level of NRK was significantly correlated with IPSS and Qmax of BPH patients. Silencing of NRK inhibited stromal cell proliferation, migration, fibrosis and EMT process, promoted apoptosis and induced cell cycle arrest, while overexpression of NRK in prostate epithelial cells showed opposite results. Meanwhile, induced fibrosis and EMT process were rescued by knockdown of NRK. Furthermore, expression level of NRK was positively correlated with that of α-SMA, collagen-I and N-cadherin, negatively correlated with that of E-cadherin.ConclusionOur novel data identified NRK was upregulated in hyperplastic prostate and associated with prostatic stromal cell proliferation, apoptosis, cell cycle, migration, fibrosis and EMT process. NRK may play important roles in the development of BPH and may be a promising therapeutic target for BPH/LUTS.

Neuroprotective mushrooms

Abstract Objectives Alternative medicines commonly supplement or, at times, replace standard medical treatment. One area of increasing attention is disease-modifying medicines for neurodegenerative diseases. However, few such alternatives have been investigated thoroughly with an eye towards understanding mechanisms of action for clinical use. Medicinal mushrooms have important health benefits and pharmacological activities with anti-inflammatory, antioxidant, antibacterial, antiviral, immunomodulatory, digestive, cytoprotective, homeostatic, and neuroprotective activities. Edible mushrooms are known to play roles in preventing age-related diseases. Several studies have revealed that polysaccharides, terpenes, and phenolic compounds are chemical components derived from mushrooms with pharmacological activities. Due to limited effective protocols for mushroom protein extraction for proteomic studies, information about these medicinally related proteins and their biological functions remains enigmatic. Methods Herein, we have performed proteomic studies of two mushroom species Laricifomes officinalis (agarikon) and Grifola frondosa (maitake). Results These studies serve to uncover a foundation for putative proteome-associated neuroprotective processes. The recovered proteins from both species show multiple cell-specific signaling pathways including unfolded protein response, and mitochondrial protein import as well as those linked to BAG2, ubiquitination, apoptosis, microautophagy, glycolysis, SNARE, and immunogenic cell signaling pathways. Conclusions This study uncovered mushroom proteome-associated proteins which serve to better understand the structural and functional properties of mushrooms used as alternative medicines for broad potential health benefits.

Screening for immune biomarkers associated with infection or protection against Ehrlichia ruminantium by RNA-sequencing analysis

Heartwater is one of the most economically important tick-borne fatal diseases of livestock. The disease is caused by the bacteria Ehrlichia ruminantium transmitted by Amblyomma ticks. Although there is evidence that interferon-gamma controls E. ruminantium growth and that cellular immune responses are protective, an effective recombinant vaccine for this disease is lacking. Analyses of markers associated with infection as well as protection will lead to a better understanding of the E. ruminantium immune response and corresponding pathways induced in sheep peripheral blood mononuclear cells (PBMC) will assist in development of such a vaccine. In this study, Biomarkers of infection (BMI) were identified as uniquely expressed genes during primary infection and biomarkers of protection (BMP) associated with immune to heartwater were identified post challenge. Sheep were experimentally infected and challenged with E. ruminantium infected ticks. The immune phenotypic and transcriptome profile of their PBMC were compared to their own naïve PBMC collected before infection. The study revealed 305 differentially expressed genes (DEGs) as BMI, of these 17 were upregulated at all three time-points investigated. These DEGs, form part of the bacterial invasion of epithelial cells Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway, and others detected from day 1 post infection and are considered predictive markers for early heartwater infection in ruminants. Similarly, a total of 332 DEGs were identified as BMP, of these 100 were upregulated and 75 were downregulated at all three time-points investigated. However, at D1PC most DEGs were downregulated (n = 1312) that correlated with a reduction in the % CD4 and CD8 T cells detected with flow cytometry. KEGG pathway analyses showed complete down regulation of T cell specific pathways possibly due to homing of immune cells to the site of infection after acquired immunity developed. At D4PC, expression levels of most of these downregulated genes increased and by D6PC they were upregulated. This indicates that the sampling time-point for biomarker analyses is important when results for acquired immune responses are inferred. This data identified DEGs that could be considered as biomarkers of protective immunity that can be used for identification of vaccine antigens and provides a strong foundation to further development of heartwater recombinant vaccines.

The glucocorticoid receptor as a master regulator of the Müller cell response to diabetic conditions in mice

Diabetic retinopathy (DR) is considered a primarily microvascular complication of diabetes. Müller glia cells are at the centre of the retinal neurovascular unit and play a critical role in DR. We therefore investigated Müller cell-specific signalling pathways that are altered in DR to identify novel targets for gene therapy. Using a multi-omics approach on purified Müller cells from diabetic db/db mice, we found the mRNA and protein expression of the glucocorticoid receptor (GR) to be significantly decreased, while its target gene cluster was down-regulated. Further, oPOSSUM TF analysis and ATAC- sequencing identified the GR as a master regulator of Müller cell response to diabetic conditions. Cortisol not only increased GR phosphorylation. It also induced changes in the expression of known GR target genes in retinal explants. Finally, retinal functionality was improved by AAV-mediated overexpression of GR in Müller cells. Our study demonstrates an important role of the glial GR in DR and implies that therapeutic approaches targeting this signalling pathway should be aimed at increasing GR expression rather than the addition of more ligand.Graphical

Structural basis for the activity and specificity of the immune checkpoint inhibitor lirilumab

The clinical success of immune checkpoint inhibitors has underscored the key role of the immune system in controlling cancer. Current FDA-approved immune checkpoint inhibitors target the regulatory receptor pathways of cytotoxic T-cells to enhance their anticancer responses. Despite an abundance of evidence that natural killer (NK) cells can also mediate potent anticancer activities, there are no FDA-approved inhibitors targeting NK cell specific checkpoint pathways. Lirilumab, the most clinically advanced NK cell checkpoint inhibitor, targets inhibitory killer immunoglobulin-like receptors (KIRs), however it has yet to conclusively demonstrate clinical efficacy. Here we describe the crystal structure of lirilumab in complex with the inhibitory KIR2DL3, revealing the precise epitope of lirilumab and the molecular mechanisms underlying KIR checkpoint blockade. Notably, the epitope includes several key amino acids that vary across the human population, and binding studies demonstrate the importance of these amino acids for lirilumab binding. These studies reveal how KIR variations in patients could influence the clinical efficacy of lirilumab and reveal general concepts for the development of immune checkpoint inhibitors targeting NK cells.

Cell and animal models of age‐related macular degeneration: Focus on oxidative stress and protein aggregation

Age‐related macular degeneration (AMD) is a multifactorial disease with a long asymptomatic development period. Due to its multifactorial feature and slow development, disease modelling capturing all the elements becomes difficult if not impossible. The disease has a strong genetic background in form of certain polymorphisms in the complement system and ARMS2/HTRA1 genes providing tools for studies regarding genetics. However, AMD is much more than genetics. At least prolonged oxidative stress and subsequent protein accumulation and aggregation together with local inflammation developing to chronic in retinal pigment epithelial (RPE) cells are the key elements of the cellular pathogenesis of AMD. The RPE cells are quiescent and responsible for the wellbeing of the entire retina functioning as the link between choroidal vasculature and other layers of the retina providing necessary support for normal retinal functions. Therefore, disturbance on the cellular homeostasis of the RPE compromise the function of the entire retina. Despite being such an integral part of the retina responsible for the vision, the RPE cells withstand one the highest levels of oxidative stress throughout a lifetime of an individual. This sets certain demands for the antioxidant system's capacity to eliminate harmful oxidant molecules and prevent the spread of oxidative injury inside the cell leading to oxidative stress. However, constant oxidative challenge leads to damages on the protective system and becomes more deleterious over time. Proteins are especially prone to oxidative damage resulting increased levels of dysfunctional or aggregating proteins challenging the repair/degradative systems of the cell. Modification of oxidative status or protein degradation systems have been shown to simulate the disease state in the RPE cells and leading to a disease resembling phenotype. In vitro models are efficient tools studying certain cell specific molecular pathways such as oxidative stress, function of antioxidant defence system, and repair/degradative pathways and discovering potential druggable targets. In vivo animal models offer information how these pathways function in a native environment and how the in vitro results are translated to a functional living organism. Also, animal models offer information about the potential administration routes as well as important pharmacological data, for example. The use of most common animals in research, rodents, needs genetic engineering, for example, to achieve desired disease modelling phenotype often lacking some critical features of the disease. However, the use of these models has greatly increased the understanding of AMD pathogenesis, lead to new discoveries on disease development later confirmed in patient population, and offered new treatment strategies.

Single-Cell RNA Sequencing Reveals Immune Cell-Specific Genes and Pathways Associated with IgA Nephropathy

Single-Cell RNA Sequencing Reveals Immune Cell-Specific Genes and Pathways Associated with IgA Nephropathy

Cell Subtypes and Cell-Specific Pathways Associated with Acute-to-Chronic Injury Transition in Posttransplant AKI

Cell Subtypes and Cell-Specific Pathways Associated with Acute-to-Chronic Injury Transition in Posttransplant AKI

Cell-specific NFIA upregulation promotes epileptogenesis by TRPV4-mediated astrocyte reactivity

BackgroundThe astrocytes in the central nervous system (CNS) exhibit morphological and functional diversity in brain region-specific pattern. Functional alterations of reactive astrocytes are commonly present in human temporal lobe epilepsy (TLE) cases, meanwhile the neuroinflammation mediated by reactive astrocytes may advance the development of hippocampal epilepsy in animal models. Nuclear factor I-A (NFIA) may regulate astrocyte diversity in the adult brain. However, whether NFIA endows the astrocytes with regional specificity to be involved in epileptogenesis remains elusive.MethodsHere, we utilize an interference RNA targeting NFIA to explore the characteristics of NFIA expression and its role in astrocyte reactivity in a 4-aminopyridine (4-AP)-induced seizure model in vivo and in vitro. Combined with the employment of a HA-tagged plasmid overexpressing NFIA, we further investigate the precise mechanisms how NIFA facilitates epileptogenesis.Results4-AP-induced NFIA upregulation in hippocampal region is astrocyte-specific, and primarily promotes detrimental actions of reactive astrocyte. In line with this phenomenon, both NFIA and vanilloid transient receptor potential 4 (TRPV4) are upregulated in hippocampal astrocytes in human samples from the TLE surgical patients and mouse samples with intraperitoneal 4-AP. NFIA directly regulates mouse astrocytic TRPV4 expression while the quantity and the functional activity of TRPV4 are required for 4-AP-induced astrocyte reactivity and release of proinflammatory cytokines in the charge of NFIA upregulation. NFIA deficiency efficiently inhibits 4-AP-induced TRPV4 upregulation, weakens astrocytic calcium activity and specific astrocyte reactivity, thereby mitigating aberrant neuronal discharges and neuronal damage, and suppressing epileptic seizure.ConclusionsOur results uncover the critical role of NFIA in astrocyte reactivity and illustrate how epileptogenic brain injury initiates cell-specific signaling pathway to dictate the astrocyte responses.

Identification of brain endothelial cell-specific genes and pathways in ischemic stroke by integrated bioinformatical analysis.

Ischemic stroke (IS) is a life-threatening condition with limited treatment options; thus, finding the potential key genes for novel therapeutic targets is urgently needed. This study aimed to explore novel candidate genes and pathways of brain microvessel endothelial cells (ECs) in IS by bioinformatics analysis. The gene expression profiles of brain tissues or brain ECs in IS mice were downloaded from the online gene expression omnibus (GEO) to obtain the differentially expressed genes (DEGs) by R software. Functional enrichment analyses were used to cluster the functions and signaling pathways of the DEGs, while DEG-associated protein-protein interaction network was performed to identify hub genes. The target microRNAs and competitive endogenous RNA networks of key hub genes were constructed by Cytoscape. Totally 84 DEGs were obtained from 6 brain tissue samples and 4 brain vascular EC samples both from IS mice in the datasets GSE74052 and GSE137482, with significant enrichment in immune responses, such as immune system processes and T-cell activation. Eight hub genes filtered by Cytoscape were validated by two other GEO datasets, wherein key genes of interest were verified by reverse transcription-polymerase chain reaction using an in vitro ischemic model of EC cultures. Our data indicated that AURKA and CENPF might be potential therapeutic target genes for IS, and Malat1/Snhg12/Xist-miR-297b-3p-CENPF, as well as Mir17 hg-miR-34b-3p-CENPF, might be RNA regulatory pathways to control IS progression. Our work identified two brain EC-specific expressed genes in IS, namely, AURKA and CENPF, as potential gene targets for IS treatment. In addition, we presented miR-297b-3p/miR-34b-3p-CENPF as the potential RNA regulatory axes to prevent pathogenesis of IS.

DNA Methylation Signatures of Multiple Sclerosis Occur Independently of Known Genetic Risk and Are Primarily Attributed to B Cells and Monocytes.

Epigenetic mechanisms can regulate how DNA is expressed independently of sequence and are known to be associated with various diseases. Among those epigenetic mechanisms, DNA methylation (DNAm) is influenced by genotype and the environment, making it an important molecular interface for studying disease etiology and progression. In this study, we examined the whole blood DNA methylation profiles of a large group of people with (pw) multiple sclerosis (MS) compared to those of controls. We reveal that methylation differences in pwMS occur independently of known genetic risk loci and show that they more strongly differentiate disease (AUC = 0.85, 95% CI 0.82-0.89, p = 1.22 × 10-29) than known genetic risk loci (AUC = 0.72, 95% CI: 0.66-0.76, p = 9.07 × 10-17). We also show that methylation differences in MS occur predominantly in B cells and monocytes and indicate the involvement of cell-specific biological pathways. Overall, this study comprehensively characterizes the immune cell-specific epigenetic architecture of MS.