Hypoxia preconditioning increases Notch1 activity by regulating DNA methylation in vitro and in vivo.

BACKGROUND: Our previous research has demonstrated that hypoxic preconditioning (HPC) can improve spatial learning and memory abilities in adult mice. Adult hippocampal neurogenesis has been associated with learning and memory. The Neurogenic locus notch homolog protein (Notch) was involved in adult hippocampal neurogenesis, as well as in learning and memory. It is currently unclear whether the Notch pathway regulates hippocampal neuroregeneration by modifying the DNA methylation status of the Notch gene following HPC. METHOD: The HPC animal model and cell model were established through repeated hypoxia exposure using mice and the mouse hippocampal neuronal cell line HT22. Step-down test was conducted on HPC mice. Real-time PCR and Western blot analysis were used to assess the mRNA and protein expression levels of Notch1 and hairy and enhancer of split1 (HES1). The presence of BrdU-positive cells and Notch1 expression in the hippocampal dental gyrus (DG) were examined with confocal microscopy. The methylation status of the Notch1 was analyzed using methylation-specific PCR (MS-PCR). HT22 cells were employed to elucidate the impact of HPC on Notch1 in vitro. RESULTS: HPC significantly improved the step-down test performance of mice with elevated levels of mRNA and protein expression of Notch1 and HES1 (P < 0.05). The intensities of the Notch1 signal in the control group, the H group and the HPC group were 2.62 ± 0.57 × 107, 2.87 ± 0.84 × 107, and 3.32 ± 0.14 × 107, respectively, and the number of BrdU (+) cells in the hippocampal DG were 1.83 ± 0.54, 3.71 ± 0.64, and 7.29 ± 0.68 respectively. Compared with that in C and H group, the intensity of the Notch1 signal and the number of BrdU (+) cells increased significantly in HPC group (P < 0.05). The methylation levels of the Notch1 promoter 0.82 ± 0.03, 0.65 ± 0.03, and 0.60 ± 0.02 in the C, H, and HPC groups, respectively. The methylation levels of Notch1 decreased significantly (P < 0.05). The effect of HPC on HT22 cells exhibited similarities to that observed in the hippocampus. CONCLUSION: HPC may confer neuroprotection by activating the Notch1 signaling pathway and regulating its methylation level, resulting in the regeneration of hippocampal neurons.

Efficient Escorting Strategy for Aggregation-Prone Notch EGF Repeats with Sparcl1.

Epidermal growth factor (EGF) repeats are present in various proteins and form well-defined structures with three disulfide bonds. One representative protein is the Notch receptor. Each EGF repeat contains unique atypical O-linked glycans, such as O-linked N-acetylglucosamine (O-GlcNAc). To generate a monoclonal antibody against the O-GlcNAc moiety in mouse Notch1, we expressed the recombinant C-terminal His6-tagged Notch1 EGF14-15 protein in HEK293T cells to prepare the immunogen. Most of the proteins were not secreted and showed higher molecular weight ladders in the cell lysate, suggesting protein aggregation. To overcome this issue, we fused Sparcl1 as an extracellular escorting tag to the N-terminus of Notch1 EGF14-15. The fusion protein was efficiently secreted extracellularly without protein aggregates in the lysates. Following PreScission protease treatment, Notch1 EGF14-15 was efficiently released from the escorting tag. Notch1 EGF14-15 prepared using this method was indeed O-GlcNAcylated. The optimal length of the escorting tag was determined by generating deletion mutants to improve the extracellular secretion of EGF14-15. Hence, a large amount of EGF14-15 was successfully prepared from the culture supernatant of HEK293T cells, which were otherwise prone to aggregation.

Upregulation of Notch Signaling and Cell-Differentiation Inhibitory Transcription Factors in Stable Chronic Obstructive Pulmonary Disease Patients.

Notch signaling is involved in the prevention of cell differentiation and cell fate in various organs, including the lungs. We aimed to determine the transcriptomic and protein expression of Notch receptors, their ligands, and related transcription factors in stable COPD. The expression and localization of Notch receptors, their ligands, and related transcription factors were measured in bronchial biopsies of individuals with stable mild/moderate (MCOPD) (n = 18) or severe/very severe (SCOPD) (n = 16) COPD, control smokers (CSs) (n = 13), and control nonsmokers (CNSs) (n = 11), and in the lung parenchyma of those with MCOPD (n = 13), CSs (n = 10), and CNSs (n = 10) using immunohistochemistry, ELISA tests, and transcriptome analyses. In the bronchial biopsies, Notch4 and HES7 significantly increased in the lamina propria of those with SCOPD compared to those with MCOPD, CSs, and CNSs. In the peripheral lung bronchiolar epithelium, Notch1 significantly increased in those with MCOPD and CSs compared to CNSs. ELISA tests of lung parenchyma homogenates showed significantly increased Notch2 in those with MCOPD compared to CSs and CNSs. Transcriptomic data in lung parenchyma showed increased DLL4 and HES1 mRNA levels in those with MCOPD and CSs compared to CNSs. These data show the increased expression of the Notch pathway in the lungs of those with stable COPD. These alterations may play a role in impairing the regenerative-reparative responses of diseased bronchioles and lung parenchyma.

Evolution and Function of the Notch Signaling Pathway: An Invertebrate Perspective.

The highly conserved Notch signaling pathway affects embryonic development, neurogenesis, homeostasis, tissue repair, immunity, and numerous other essential processes. Although previous studies have demonstrated the location and function of the core components of Notch signaling in various animal phyla, a more comprehensive summary of the Notch core components in lower organisms is still required. In this review, we objectively summarize the molecular features of the Notch signaling pathway constituents, their current expression profiles, and their functions in invertebrates, with emphasis on their effects on neurogenesis and regeneration. We also analyze the evolution and other facets of Notch signaling and hope that the contents of this review will be useful to interested researchers.

Force-free activation of Notch with DNA origami.

The Notch signaling pathway is a highly conserved, fundamental process to embryogenesis and neurogenesis. While force-induced conformational change is known to activate Notch receptors, Smyrlaki et al. recently used DNA origami to reveal an additional, force-independent mode of Notch activation via soluble presentation of spatially controlled ligand nanopatterns.

Unraveling paralog-specific Notch signaling through thermodynamics of ternary complex formation and transcriptional activation of chimeric receptors.

Notch signaling in humans is mediated by four paralogous receptors that share conserved architectures and possess overlapping, yet non-redundant functions. The receptors share a canonical activation pathway wherein upon extracellular ligand binding, the Notch intracellular domain (NICD) is cleaved from the membrane and translocates to the nucleus where its N-terminal RBP-j-associated molecule (RAM) region and ankyrin repeat (ANK) domain bind transcription factor CSL and recruit co-activator Mastermind-like-1 (MAML1) to activate transcription. However, different paralogs can lead to distinct outcomes. To better understand paralog-specific differences in Notch signaling, we performed a thermodynamic analysis of the Notch transcriptional activation complexes for all four Notch paralogs using isothermal titration calorimetry. Using chimeric constructs, we find that the RAM region is the primary determinant of stability of binary RAMANK:CSL complexes, and that the ANK regions are largely the determinants of MAML1 binding to pre-formed RAMANK:CSL complexes. Free energies of these binding reactions (ΔGRA and ΔGMAML) vary among the four Notch paralogs, although variations for Notch2, 3, and 4 offset in the free energy of the ternary complex (ΔGTC, where ΔGTC = ΔGRA + ΔGMAML). To probe how these affinity differences affect Notch signaling, we performed transcriptional activation assays with the paralogous and chimeric NICDs, and analyzed the results with an independent multiplicative model that quantifies contributions of the paralogous RAM, ANK, and C-terminal regions (CTR) to activation. This analysis shows that transcription activation correlates with ΔGTC, but that activation is further modified by CTR identity in a paralog-specific way.

Identifications of three novel alleles of Serrate in Drosophila.

The Notch signaling pathway, an evolutionarily highly conserved pathway, participates in various essential physiological processes in organisms. Activation of Notch signaling in the canonical manner requires the combination of ligand and receptor. There are two ligands of Notch in Drosophila: Delta (Dl) and Serrate (Ser). A mutation mf157 is identified for causing nicks of fly wings in genetic analysis from a mutant library (unpublished) that was established previously. Immunofluorescent staining illustrates that mf157 represses the expression of Cut and Wingless (Wg), the targets of Notch signaling. MARCM cloning analysis reveals that mf157 functions at the same level or the upstream of ligands of Notch in signaling sending cells. Sequencing demonstrates that mf157 is a novel allele of the Ser gene. Subsequently, mf553 and mf167 are also identified as new alleles of Ser from our library. Furthermore, the complementary assays and the examination of transcripts confirm the sequencing results. Besides, the repressed phenotypes of Notch signaling were reverted by transposon excision experiments of mf157. In conclusion, we identify three fresh alleles of Ser. Our works supply additional genetic resources for further study of functions of Ser and Notch signaling regulation.

Corpora cavernosa fibroblasts mediate penile erection.

Penile erection is mediated by the corpora cavernosa, a trabecular-like vascular bed that enlarges upon vasodilation, but its regulation is not completely understood. Here, we show that perivascular fibroblasts in the corpora cavernosa support vasodilation by reducing norepinephrine availability. The effect on penile blood flow depends on the number of fibroblasts, which is regulated by erectile activity. Erection dynamically alters the positional arrangement of fibroblasts, temporarily down-regulating Notch signaling. Inhibition of Notch increases fibroblast numbers and consequently raises penile blood flow. Continuous Notch activation lowers fibroblast numbers and reduces penile blood perfusion. Recurrent erections stimulate fibroblast proliferation and limit vasoconstriction, whereas aging reduces the number of fibroblasts and lowers penile blood flow. Our findings reveal adaptive, erectile activity-dependent modulation of penile blood flow by fibroblasts.

Protein aggregates containing wild-type and mutant NOTCH3 are major drivers of arterial pathology in CADASIL.

Loss of arterial smooth muscle cells (SMCs) and abnormal accumulation of the extracellular domain of the NOTCH3 receptor (Notch3ECD) are the 2 core features of CADASIL, a common cerebral small vessel disease caused by highly stereotyped dominant mutations in NOTCH3. Yet the relationship between NOTCH3 receptor activity, Notch3ECD accumulation, and arterial SMC loss has remained elusive, hampering the development of disease-modifying therapies. Using dedicated histopathological and multiscale imaging modalities, we could detect and quantify previously undetectable CADASIL-driven arterial SMC loss in the CNS of mice expressing the archetypal Arg169Cys mutation. We found that arterial pathology was more severe and Notch3ECD accumulation greater in transgenic mice overexpressing the mutation on a wild-type Notch3 background (TgNotch3R169C) than in knockin Notch3R170C/R170C mice expressing this mutation without a wild-type Notch3 copy. Notably, expression of Notch3-regulated genes was essentially unchanged in TgNotch3R169C arteries. We further showed that wild-type Notch3ECD coaggregated with mutant Notch3ECD and that elimination of 1 copy of wild-type Notch3 in TgNotch3R169C was sufficient to attenuate Notch3ECD accumulation and arterial pathology. These findings suggest that Notch3ECD accumulation, involving mutant and wild-type NOTCH3, is a major driver of arterial SMC loss in CADASIL, paving the way for NOTCH3-lowering therapeutic strategies.

Characterization of galactosyltransferase and sialyltransferase genes mediating the elongation of the extracellular O-GlcNAc glycans.

O-GlcNAc is a unique post-translational modification found in cytoplasmic, nuclear, and mitochondrial proteins. In a limited number of extracellular proteins, O-GlcNAc modifications occur through the action of EOGT, which specifically modifies subsets of epidermal growth factor-like (EGF) domain-containing proteins such as Notch receptors. The abnormalities due to EOGT mutations in mice and humans and the increased EOGT expression in several cancers signify the importance of EOGT pathophysiology and extracellular O-GlcNAc. Unlike intracellular O-GlcNAc monosaccharides, extracellular O-GlcNAc extends to form elongated glycan structures. However, the enzymes involved in the O-GlcNAc glycan extension have not yet been reported. In our study, we comprehensively screened potential galactosyltransferase and sialyltransferase genes related to the canonical O-GlcNAc glycan pathway and revealed the essential roles of B4GALT1 and ST3GAL4 in O-GlcNAc glycan elongation in human HEK293 cells. These findings were confirmed by sequential glycosylation of Drosophila EGF20 in vitro by EOGT, ß4GalT-1, and ST3Gal-IV. Thus, the findings from our study throw light on the specific glycosyltransferases that mediate O-GlcNAc glycan elongation in human HEK293 cells.

Notch signaling and targeted therapy in non-small cell lung cancer.

The Notch signaling pathway plays pivotal roles in cell proliferation, stemness and invasion of non-small cell lung cancer (NSCLC). The human Notch family consists of four receptors, namely Notch1, Notch2, Notch3, and Notch4. These receptors are transmembrane proteins that play crucial roles in various cellular processes. Notch1 mostly acts as a pro-carcinogenic factor in NSCLC but sometimes acts as a suppressor. Notch2 has been demonstrated to inhibit the growth and progression of NSCLC, whereas Notch3 facilitates these biological behaviors of NSCLC. The role of Notch4 in NSCLC has not been fully elucidated, but it is evident that Notch4 promotes tumor progression. At present, drugs targeting the Notch pathway are being explored for NSCLC therapy, a majority of which are already in the stage of preclinical research and clinical trials, with bright prospects in the clinical treatment of NSCLC.

Alternative mechanisms of Notch activation by partitioning into distinct endosomal domains.

Different membrane microdomain compositions provide unique environments that can regulate signaling receptor function. We identify microdomains on the endosome membrane of Drosophila endosomes, enriched in lipid-raft or clathrin/ESCRT-0, which are associated with Notch activation by distinct, ligand-independent mechanisms. Transfer of Notch between microdomains is regulated by Deltex and Suppressor of deltex ubiquitin ligases and is limited by a gate-keeper role for ESCRT complexes. Ubiquitination of Notch by Deltex recruits it to the clathrin/ESCRT-0 microdomain and enhances Notch activation by an ADAM10-independent/TRPML-dependent mechanism. This requirement for Deltex is bypassed by the downregulation of ESCRT-III. In contrast, while ESCRT-I depletion also activates Notch, it does so by an ADAM10-dependent/TRPML-independent mechanism and Notch is retained in the lipid raft-like microdomain. In the absence of such endosomal perturbation, different activating Notch mutations also localize to different microdomains and are activated by different mechanisms. Our findings demonstrate the interplay between Notch regulators, endosomal trafficking components, and Notch genetics, which defines membrane locations and activation mechanisms.

Akt3 activation by R-Ras in an endothelial cell enforces quiescence and barrier stability of neighboring endothelial cells via Jagged1.

Communication between adjacent endothelial cells is important for the homeostasis of blood vessels. We show that quiescent endothelial cells use Jagged1 to instruct neighboring endothelial cells to assume a quiescent phenotype and secure the endothelial barrier. This phenotype enforcement by neighboring cells is operated by R-Ras through activation of Akt3, which results in upregulation of a Notch ligand Jagged1 and consequential upregulation of Notch target genes, such as UNC5B, and VE-cadherin accumulation in the neighboring cells. These signaling events lead to the stable interaction between neighboring endothelial cells to continue to fortify juxtacrine signaling via Jagged1-Notch. This mode of intercellular signaling provides a positive feedback regulation of endothelial cell-cell interactions and cellular quiescence required for the stabilization of the endothelium.

Notch intracellular domains form transcriptionally active heterodimeric complexes on sequence-paired sites.

Notch signaling is universally conserved in metazoans where it is important for a wide variety of both normal and abnormal physiology. All four mammalian Notch receptors are activated by a conserved mechanism that releases Notch intracellular domains (NICDs) from the plasma membrane to translocate to the nucleus. Once there, NICDs interact through highly conserved ankyrin domains to form head-to-head homodimers on Notch sensitive promoters and stimulate transcription. Due to the highly conserved nature of these Notch ankyrin domains in all four mammalian Notch proteins, we hypothesized that NICDs may also engage in heterodimerization. Our results reveal the presence of two NICD dimerization states that can both engage in homo and heterodimerization. Using a Co-IP approach, we show that all NICD's can form non-transcriptionally active dimers and that the N4ICD appears to perform this function better than the other NICDs. Using a combination of ChIP analysis and transcriptional reporter assays, we also demonstrate the formation of transcriptionally active heterodimers that form on DNA. In particular, we demonstrate heterodimerization between the N2ICD and N4ICD and show that this heterodimer pair appears to exhibit differential activity on various Notch sensitive promoters. These results illustrate a new diversification of Notch signaling mechanisms which will help us better understand basic Notch function.

Notch signaling and Bsh homeodomain activity are integrated to diversify Drosophila lamina neuron types.

Notch signaling is an evolutionarily conserved pathway for specifying binary neuronal fates, yet how it specifies different fates in different contexts remains elusive. In our accompanying paper, using the Drosophila lamina neuron types (L1-L5) as a model, we show that the primary homeodomain transcription factor (HDTF) Bsh activates secondary HDTFs Ap (L4) and Pdm3 (L5) and specifies L4/L5 neuronal fates. Here we test the hypothesis that Notch signaling enables Bsh to differentially specify L4 and L5 fates. We show asymmetric Notch signaling between newborn L4 and L5 neurons, but they are not siblings; rather, Notch signaling in L4 is due to Delta expression in adjacent L1 neurons. While Notch signaling and Bsh expression are mutually independent, Notch is necessary and sufficient for Bsh to specify L4 fate over L5. The NotchON L4, compared to NotchOFF L5, has a distinct open chromatin landscape which allows Bsh to bind distinct genomic loci, leading to L4-specific identity gene transcription. We propose a novel model in which Notch signaling is integrated with the primary HDTF activity to diversify neuron types by directly or indirectly generating a distinct open chromatin landscape that constrains the pool of genes that a primary HDTF can activate.

A novel search engine for proteins involved in Notch crosstalk signaling pathways.

To regulate biological activity in humans, the Notch signaling pathway (NSP) plays an essential role in a wide array of cellular development and differentiation process. In recent years, many studies have reported that aberrant activation of Notch is associated with the tumor process; but no appropriate database exists to fill this significant gap. To address this, we created a pioneering database NCSp, which is open access and comprises intercommunicating pathways and related protein mutations. This allows scientists to understand better the cause of single amino acid mutations in proteins. Therefore, NCSp provides information on the predicted functional effect of human protein mutations, which aids in understanding the importance of mutations linked to the Notch crosstalk signaling pathways in cancerous and non-cancerous systems. This database might be helpful for therapeutic mutation analysis, molecular biology, and structural biology researchers. The NCSp database can be accessed through https://bioserver3.physics.iisc.ac.in/cgi-bin/nccspd/.

Disparate macrophage responses are linked to infection outcome of Hantan virus in humans or rodents.

Hantaan virus (HTNV) is asymptomatically carried by rodents, yet causes lethal hemorrhagic fever with renal syndrome in humans, the underlying mechanisms of which remain to be elucidated. Here, we show that differential macrophage responses may determine disparate infection outcomes. In mice, late-phase inactivation of inflammatory macrophage prevents cytokine storm syndrome that usually occurs in HTNV-infected patients. This is attained by elaborate crosstalk between Notch and NF-κB pathways. Mechanistically, Notch receptors activated by HTNV enhance NF-κB signaling by recruiting IKKß and p65, promoting inflammatory macrophage polarization in both species. However, in mice rather than humans, Notch-mediated inflammation is timely restrained by a series of murine-specific long noncoding RNAs transcribed by the Notch pathway in a negative feedback manner. Among them, the lnc-ip65 detaches p65 from the Notch receptor and inhibits p65 phosphorylation, rewiring macrophages from the pro-inflammation to the pro-resolution phenotype. Genetic ablation of lnc-ip65 leads to destructive HTNV infection in mice. Thus, our findings reveal an immune-braking function of murine noncoding RNAs, offering a special therapeutic strategy for HTNV infection.

Exploring the Multiple Roles of Notch1 in Biological Development: An Analysis and Study Based on Phylogenetics and Transcriptomics.

At present, there is a research gap concerning the specific functions and mechanisms of the Notch gene family and its signaling pathway in jawless vertebrates. In this study, we identified a Notch1 homologue (Lr. Notch1) in the Lethenteron reissneri database. Through bioinformatics analysis, we identified Lr. Notch1 as the likely common ancestor gene of the Notch gene family in higher vertebrates, indicating a high degree of conservation in the Notch gene family and its signaling pathways. To validate the biological function of Lr. Notch1, we conducted targeted silencing of Lr. Notch1 in L. reissneri and analyzed the resultant gene expression profile before and after silencing using transcriptome analysis. Our findings revealed that the silencing of Lr. Notch1 resulted in differential expression of pathways and genes associated with signal transduction, immune regulation, and metabolic regulation, mirroring the biological function of the Notch signaling pathway in higher vertebrates. This article systematically elucidated the origin and evolution of the Notch gene family while also validating the biological function of Lr. Notch1. These insights offer valuable clues for understanding the evolution of the Notch signaling pathway and establish a foundation for future research on the origin of the Notch signaling pathway, as well as its implications in human diseases and immunomodulation.

Pudgy mouse rib deformities emanate from abnormal paravertebral longitudinal cartilage/bone accumulations.

The pudgy (pu/pu) mouse, caused by a recessive mutation in the Notch family Delta like-3 gene (Dll3), has severe rib, vertebral body and intervertebral disc abnormalities. Using whole-mount preparations and serial histologic sections we demonstrate: 1) localized paravertebral longitudinal cartilage/bone accumulations (PVLC/BAs) invariably associated with branched, fused and asymmetrically spaced ribs that emanate from it laterally; 2) abnormal rib formation immediately adjacent to abnormal vertebral body and intervertebral disc formation in asymmetric right/left fashion; and 3) patterns of rib deformation that differ in each mouse. Normal BALB/c embryo and age-matched non-affected pu/+ mice assessments allow for pu/pu comparisons. The Dll3 Notch family gene is involved in normal somitogenesis via the segmentation clock mechanism. Although pathogenesis of rib deformation is initially triggered by the Dll3 gene mutation, these findings of abnormal asymmetric costo-vertebral region structure imply that differing patterns cannot be attributed to this single gene mutation alone. All findings implicate a dual mechanism of malformation: the Dll3 gene mutation leading to subtle timing differences in traveling oscillation waves of the segmentation clock and further subsequent misdirection of tissue formation by altered chemical reaction-diffusion and epigenetic landscape responses. PVLC/BAs appear as primary supramolecular structures underlying severe rib malformation associated both with time-sensitive segmentation clock mutations and subsequent reactions.

The role of the Notch signaling pathway in bacterial infectious diseases.

The Notch signaling pathway is the most crucial link in the normal operation and maintenance of physiological functions of mammalian life processes. Notch receptors interact with ligands and this leads to three cleavages and goes on to enter the nucleus to initiate the transcription of target genes. The Notch signaling pathway deeply participates in the differentiation and function of various cells, including immune cells. Recent studies indicate that the outcomes of Notch signaling are changeable and highly dependent on different bacterial infection. The Notch signaling pathway plays a different role in promoting and inhibiting bacterial infection. In this review, we focus on the latest research findings of the Notch signaling pathway in bacterial infectious diseases. The Notch signaling pathway is critically involved in a variety of development processes of immunosuppression of different APCs. The Notch signaling pathway leads to functional changes in epithelial cells to aggravate tissue damage. Specifically, we illustrate the regulatory mechanism of the Notch signaling pathway in various bacterial infections, such as Mycobacterium tuberculosis, Mycobacterium avium paratuberculosis, Mycobacterium leprae, Helicobacter pylori, Klebsiella pneumoniae, Bacillus subtilis, Staphylococcus aureus, Ehrlichia chaffeensis and sepsis. Collectively, this review will not only help beginners intuitively and systematically understand the Notch signaling pathway in bacterial infectious diseases but also help experts to generate fresh insight in this field.