Comparative Analysis of Transcription Factors TWIST2, GATA3, and HES5 in Glioblastoma Multiforme : Evaluating Biomarker Potential and Therapeutic Targets Using In Silico Methods.

Objective: Glioblastoma multiforme (GBM) is characterized by substantial heterogeneity and limited therapeutic options. As molecular approaches to central nervous system (CNS) tumors have gained prominence, this study examined the roles of three genes, TWIST2, GATA3, and HES5, known to be involved in oncogenesis, developmental processes, and maintenance of cancer stem cell properties, which have not yet been extensively studied in GBM. This study is the first to present gene expression data for TWIST2, GATA3, and HES5 specifically within the context of GBM patient survival. Methods: Gene expression data for TWIST2, GATA3, and HES5 were collected from GBM and normal brain tissues using datasets from The Cancer Genome Atlas (TCGA) via the Genomic Data Commons (GDC) portal and the Genotype-Tissue Expression (GTEx) database. These data were rigorously analyzed using in silico methods. Results: All three genes were significantly more expressed in GBM tissues than in normal tissues. TWIST2 and GATA3 were linked to lower survival rates in GBM patients. Interestingly, higher HES5 levels were associated with better survival rates, suggesting a complex role that needs more investigation. Conclusion: This study shows that TWIST2, GATA3, and HES5 could help predict outcomes in GBM patients. Our multigene model offers a better understanding of GBM and points to new treatment options, bringing hope for improved therapies and patient outcomes. This research advances our knowledge of GBM and highlights the potential of molecular diagnostics in oncology.

NOTCH3 and Pulmonary Arterial Hypertension.

NOTCH3 receptor signaling has been linked to the regulation of smooth muscle cell proliferation and the maintenance of smooth muscle cells in an undifferentiated state. Pulmonary arterial hypertension (World Health Organization Group 1 idiopathic disease: PAH) is a fatal disease characterized clinically by elevated pulmonary vascular resistance caused by extensive vascular smooth muscle cell proliferation, perivascular inflammation, and asymmetric neointimal hyperplasia in precapillary pulmonary arteries. In this review, a detailed overview of the specific role of NOTCH3 signaling in PAH, including its mechanisms of activation by a select ligand, downstream signaling effectors, and physiologic effects within the pulmonary vascular tree, is provided. Animal models showing the importance of the NOTCH3 pathway in clinical PAH will be discussed. New drugs and biologics that inhibit NOTCH3 signaling and reverse this deadly disease are highlighted.

Laminar-selective spinal astrocyte population capable of converting tactile information into nociceptive in rats.

We previously identified a spinal astrocyte population that expresses hairy and enhancer of split 5 (Hes5) and is selectively present in superficial laminae in mice. However, it was unclear whether such astrocyte heterogeneity is commonly observed across species. Using adeno-associated viral (AAV) vectors incorporating a rat Hes5 promotor (AAV-Hes5P), we found that AAV-Hes5P-captured astrocytes were selectively located in the superficial laminae in rats. Furthermore, activation of AAV-Hes5P+ astrocytes elicited allodynia-like behavior and increased c-FOS+ cells in the superficial laminae. Thus, laminar-selective Hes5+ astrocytes are conserved beyond species and have the capability to convert tactile information to nociceptive.

Effects of Subchronic Propofol Administration on the Proliferation and Differentiation of Neural Stem Cells in Rat Hippocampus.

Background: Although controversial, experimental data suggest the use of propofol may be associated with neurotoxicity. The mechanisms responsible for propofol neurotoxicity in animals are not yet clear. Objective: This study aimed to determine the effects of propofol on the proliferation of neural stem cells in rat hippocampus and the mechanisms underlying these effects. Methods: Forty-five adult male Sprague-Dawley rats were randomly divided into 5 groups: Control (N group), intralipid (V group), 30 mg/kg propofol (Prop30 group), 60 mg/kg propofol (Prop60 group), and 120 mg/kg propofol (Prop120 group). The rats in all groups received 5, once daily intraperitoneal injections. For each of the 5 days, the N group received 6 mL/kg normal saline, the V group received 6 mL/kg fat emulsion, the Prop30 group received 30 mg/kg propofol, the Prop60 group received 60 mg/kg propofol, and the Prop120 group received 120 mg/kg propofol. Memory function was scored daily using the Morris water maze test. Immunofluorescence staining was used to histologically monitor the proliferation and differentiation of the rats' hippocampal neural stem cells, and real time quantitative polymerase chain reaction and Western blotting were used to determine the expression of Notch3, Hes1, and Hes5. Results: Compared with the N group, the Prop120 group exhibited reduced learning and memory, whereas there were no significant differences for the Prop60 group. The number of ß-tubulin III+ cells increased in the Prop60 group, but decreased in the Prop120 group. Compared with the N group, the relative expression of Notch3 and Hes5 increased significantly in the Prop60 group, whereas this expression decreased in the Prop120 group. Conclusions: These data demonstrate that repeated, subchronic (5 days) intraperitoneal injections of 60 mg/kg propofol can effectively promote rat hippocampal neural stem cells proliferation and differentiation, and that this is likely mediated by its effects on the Notch3-Hes5 pathway.

Transcription factor SOX15 regulates stem cell pluripotency and promotes neural fate during differentiation by activating the neurogenic gene Hes5.

SOX2 and SOX15 are Sox family transcription factors enriched in embryonic stem cells (ESCs). The role of SOX2 in activating gene expression programs essential for stem cell self-renewal and acquisition of pluripotency during somatic cell reprogramming is well-documented. However, the contribution of SOX15 to these processes is unclear and often presumed redundant with SOX2 largely because overexpression of SOX15 can partially restore self-renewal in SOX2-deficient ESCs. Here, we show that SOX15 contributes to stem cell maintenance by cooperating with ESC-enriched transcriptional coactivators to ensure optimal expression of pluripotency-associated genes. We demonstrate that SOX15 depletion compromises reprogramming of fibroblasts to pluripotency which cannot be compensated by SOX2. Ectopic expression of SOX15 promotes the reversion of a postimplantation, epiblast stem cell state back to a preimplantation, ESC-like identity even though SOX2 is expressed in both cell states. We also uncover a role of SOX15 in lineage specification, by showing that loss of SOX15 leads to defects in commitment of ESCs to neural fates. SOX15 promotes neural differentiation by binding to and activating a previously uncharacterized distal enhancer of a key neurogenic regulator, Hes5. Together, these findings identify a multifaceted role of SOX15 in induction and maintenance of pluripotency and neural differentiation.

Effects of hyperbaric oxygen on Notch signaling pathway after severe carbon monoxide poisoning in mice.

Demyelination of the cerebral white matter is the most common pathological change after carbon monoxide (CO) poisoning. Notch signaling, the mechanism underlying the differentiation of astrocytes and oligodendrocytes, is critical to remyelination of the white matter after brain lesion. The purpose of this work was to determine the effects of hyperbaric oxygen (HBO) on Notch signaling pathway after CO poisoning for the explanation of the protective effects of HBO on CO-poisoning-related cerebral white matter demyelination. The male C57 BL/6 mice with severe CO poisoning were treated by HBO. And HBO therapy shortened the escape latency and improved the body mass after CO poisoning. HBO therapy also significantly suppressed protein and mRNA levels of Notch1 and Hes5 after CO poisoning. Our findings suggested that HBO could suppress the activation of Notch signaling pathway after CO poisoning, which is the mechanism underlying the neuroprotection of HBO on demyelination after severe CO poisoning.

Dynamic switching of lateral inhibition spatial patterns.

Hes genes are transcriptional repressors activated by Notch. In the developing mouse neural tissue, HES5 expression oscillates in neural progenitors (Manning et al. 2019 Nat. Commun. 10, 1-19 (doi:10.1038/s41467-019-10734-8)) and is spatially organized in small clusters of cells with synchronized expression (microclusters). Furthermore, these microclusters are arranged with a spatial periodicity of three-four cells in the dorso-ventral axis and show regular switching between HES5 high/low expression on a longer time scale and larger amplitude than individual temporal oscillators (Biga et al. 2021 Mol. Syst. Biol. 17, e9902 (doi:10.15252/msb.20209902)). However, our initial computational modelling of coupled HES5 could not explain these features of the experimental data. In this study, we provide theoretical results that address these issues with biologically pertinent additions. Here, we report that extending Notch signalling to non-neighbouring progenitor cells is sufficient to generate spatial periodicity of the correct size. In addition, introducing a regular perturbation of Notch signalling by the emerging differentiating cells induces a temporal switching in the spatial pattern, which is longer than an individual cell's periodicity. Thus, with these two new mechanisms, a computational model delivers outputs that closely resemble the complex tissue-level HES5 dynamics. Finally, we predict that such dynamic patterning spreads out differentiation events in space, complementing our previous findings whereby the local synchronization controls the rate of differentiation.

African swine fever virus protein MGF-505-7R promotes virulence and pathogenesis by inhibiting JAK1- and JAK2-mediated signaling.

African swine fever virus (ASFV) is a large DNA virus that is highly contagious and pathogenic in domestic pigs with a mortality rate up to 100%. However, how ASFV suppresses JAK-STAT1 signaling to evade the immune response remains unclear. In this study, we found that the ASFV-encoded protein MGF-505-7R inhibited proinflammatory IFN-γ-mediated JAK-STAT1 signaling. Mechanistically, MGF-505-7R was found to interact with JAK1 and JAK2 and mediate their degradation. Further study indicated that MGF-505-7R promoted degradation of JAK1 and JAK2 by upregulating the E3 ubiquitin ligase RNF125 expression and inhibiting expression of Hes5, respectively. Consistently, MGF-505-7R-deficient ASFV induced high levels of IRF1 expression and displayed compromised replication both in primary porcine alveolar macrophages and pigs compared with wild-type ASFV. Furthermore, MGF-505-7R deficiency attenuated the virulence of the ASFV and pathogenesis of ASF in pigs. These findings suggest that the JAK-STAT1 axis mediates the innate immune response to the ASFV and that MGF-505-7R plays a critical role in the virulence of the ASFV and pathogenesis of ASF by antagonizing this axis. Thus, we conclude that deletion of MGF-505-7R may serve as a strategy to develop attenuated vaccines against the ASFV.

Notch1 and Galectin-3 Modulate Cortical Reactive Astrocyte Response After Brain Injury.

After a brain lesion, highly specialized cortical astrocytes react, supporting the closure or replacement of the damaged tissue, but fail to regulate neural plasticity. Growing evidence indicates that repair response leads astrocytes to reprogram, acquiring a partially restricted regenerative phenotype in vivo and neural stem cells (NSC) hallmarks in vitro. However, the molecular factors involved in astrocyte reactivity, the reparative response, and their relation to adult neurogenesis are poorly understood and remain an area of intense investigation in regenerative medicine. In this context, we addressed the role of Notch1 signaling and the effect of Galectin-3 (Gal3) as underlying molecular candidates involved in cortical astrocyte response to injury. Notch signaling is part of a specific neurogenic microenvironment that maintains NSC and neural progenitors, and Gal3 has a preferential spatial distribution across the cortex and has a central role in the proliferative capacity of reactive astrocytes. We report that in vitro scratch-reactivated cortical astrocytes from C57Bl/6J neonatal mice present nuclear Notch1 intracellular domain (NICD1), indicating Notch1 activation. Colocalization analysis revealed a subpopulation of reactive astrocytes at the lesion border with colocalized NICD1/Jagged1 complexes compared with astrocytes located far from the border. Moreover, we found that Gal3 increased intracellularly, in contrast to its extracellular localization in non-reactive astrocytes, and NICD1/Gal3 pattern distribution shifted from diffuse to vesicular upon astrocyte reactivation. In vitro, Gal3-/- reactive astrocytes showed abolished Notch1 signaling at the lesion core. Notch1 receptor, its ligands (Jagged1 and Delta-like1), and Hes5 target gene were upregulated in C57Bl/6J reactive astrocytes, but not in Gal3-/- reactive astrocytes. Finally, we report that Gal3-/- mice submitted to a traumatic brain injury model in the somatosensory cortex presented a disrupted response characterized by the reduced number of GFAP reactive astrocytes, with smaller cell body perimeter and decreased NICD1 presence at the lesion core. These results suggest that Gal3 might be essential to the proper activation of Notch signaling, facilitating the cleavage of Notch1 and nuclear translocation of NICD1 into the nucleus of reactive cortical astrocytes. Additionally, we hypothesize that reactive astrocyte response could be dependent on Notch1/Jagged1-Hes5 signaling activation following brain injury.

A dynamic, spatially periodic, micro-pattern of HES5 underlies neurogenesis in the mouse spinal cord.

Ultradian oscillations of HES Transcription Factors (TFs) at the single-cell level enable cell state transitions. However, the tissue-level organisation of HES5 dynamics in neurogenesis is unknown. Here, we analyse the expression of HES5 ex vivo in the developing mouse ventral spinal cord and identify microclusters of 4-6 cells with positively correlated HES5 level and ultradian dynamics. These microclusters are spatially periodic along the dorsoventral axis and temporally dynamic, alternating between high and low expression with a supra-ultradian persistence time. We show that Notch signalling is required for temporal dynamics but not the spatial periodicity of HES5. Few Neurogenin 2 cells are observed per cluster, irrespective of high or low state, suggesting that the microcluster organisation of HES5 enables the stable selection of differentiating cells. Computational modelling predicts that different cell coupling strengths underlie the HES5 spatial patterns and rate of differentiation, which is consistent with comparison between the motoneuron and interneuron progenitor domains. Our work shows a previously unrecognised spatiotemporal organisation of neurogenesis, emergent at the tissue level from the synthesis of single-cell dynamics.

Notch intracellular domain regulates glioblastoma proliferation through the Notch1 signaling pathway.

Notch intracellular domain (NICD), also known as the activated form of Notch1 is closely associated with cell differentiation and tumor invasion. However, the role of NICD in glioblastoma (GBM) proliferation and the underlying regulatory mechanism remains unclear. The present study aimed to investigate the expression of NICD and Notch1 downstream gene HES5 in human GBM and normal brain samples and to further detect the effect of NICD on human GBM cell proliferation. For this purpose, western blotting and immunohistochemical staining were performed to analyze the expression of NICD in human GBM tissues, while western blotting and reverse-transcription quantitative PCR experiments were used to analyze the expression of Hes5 in human GBM tissues. A Flag-NICD vector was used to overexpress NICD in U87 cells and compound E and small interfering (si) Notch1 were used to downregulate NICD. Cellular proliferation curves were generated and BrdU assays performed to evaluate the proliferation of U87 cells. The results demonstrated that compared with normal brain tissues, the level of NICD protein in human GBM tissues was upregulated and the protein and mRNA levels of Hes5 were also upregulated in GBM tissues indicating that the Notch1 signaling pathway is activated in GBM. Overexpression of NICD promoted the proliferation of U87 cells in vitro while downregulation of NICD by treatment with compound E or siNotch1 suppressed the proliferation of U87 cells in vitro. In conclusion, NICD was upregulated in human GBM and NICD promoted GBM proliferation via the Notch1 signaling pathway. NICD may be a potential diagnostic marker and therapeutic target for GBM treatment.

Aryl Hydrocarbon Receptor is Essential in the Control of Lung Club Cell Homeostasis.

BACKGROUND: Club cells play an important role in maintaining lung homeostasis and aryl hydrocarbon receptor (AhR) is known to be important in xenobiotic metabolism, but its role in regulating club cells is currently unknown. METHODS: To this end, mice with club cell-specific AhR deficiency were generated and evaluated in a model of antigen (ovalbumin, OVA)-induced airway inflammation for the number of infiltrating inflammatory cells, the levels of cytokines and CC10 and Notch signaling by standard methods. RESULTS: After OVA sensitization and challenge, Scgb1a1-Cre; Ahrflox/flox mice showed aggravated levels of pulmonary inflammation with increased levels of inflammatory cells and cytokines 1 day after challenge as compared to those seen in their littermate controls, but in contrast to the littermate controls, no significant change in the levels of CC10 and SP-D was noted in Scgb1a1-Cre; Ahrflox/flox mice. Surprisingly, 7 days after the challenge, while, as expected, wild-type mice recovered from acute inflammation, significantly increased lymphocytic infiltration was noted in Scgb1a1-Cre; Ahrflox/flox mice, suggesting their defective mechanism of recovery. Mechanistically, this was due, in part, to the decreased Notch1 signaling and expression of its downstream gene, HES5, while AhR was shown to positively regulate Notch1 expression via its transactivating activity targeting the xenobiotic response element in the promoter region of Notch1 gene. CONCLUSION: Under the condition of pulmonary inflammation, AhR is critical in controlling lung club cell homeostasis via targeting Notch1 signaling and the generation of anti-inflammatory mediators.

A transient decrease in mitochondrial activity contributes to establish the ganglion cell fate in retina adapted for high acuity vision.

Although the plan of the retina is well conserved in vertebrates, there are considerable variations in cell type diversity and number, as well as in the organization and properties of the tissue. The high ratios of retinal ganglion cells (RGCs) to cones in primate fovea and bird retinas favor neural circuits essential for high visual acuity and color vision. The role that cell metabolism could play in cell fate decision during embryonic development of the nervous system is still largely unknown. Here, we describe how subtle changes of mitochondrial activity along the pathway converting uncommitted progenitors into newborn RGCs increase the recruitment of RGC-fated progenitors. ATOH7, a proneural protein dedicated to the production of RGCs in vertebrates, activates transcription of the Hes5.3 gene in pre-committed progenitors. The HES5.3 protein, in turn, regulates a transient decrease in mitochondrial activity via the retinoic acid signaling pathway few hours before cell commitment. This metabolic shift lengthens the progression of the ultimate cell cycle and is a necessary step for upregulating Atoh7 and promoting RGC differentiation.

Hes5.9 Coordinate FGF and Notch Signaling to Modulate Gastrulation via Regulating Cell Fate Specification and Cell Migration in Xenopus tropicalis.

Gastrulation drives the establishment of three germ layers and embryonic axes during frog embryonic development. Mesodermal cell fate specification and morphogenetic movements are vital factors coordinating gastrulation, which are regulated by numerous signaling pathways, such as the Wnt (Wingless/Integrated), Notch, and FGF (Fibroblast growth factor) pathways. However, the coordination of the Notch and FGF signaling pathways during gastrulation remains unclear. We identified a novel helix-loop-helix DNA binding domain gene (Hes5.9), which was regulated by the FGF and Notch signaling pathways during gastrulation. Furthermore, gain- and loss-of-function of Hes5.9 led to defective cell migration and disturbed the expression patterns of mesodermal and endodermal marker genes, thus interfering with gastrulation. Collectively, these results suggest that Hes5.9 plays a crucial role in cell fate decisions and cell migration during gastrulation, which is modulated by the FGF and Notch signaling pathways.

Effects of Notch signaling components from breast cancer cells treated in culture with resveratrol.

Resveratrol (Res) has an anti-tumor effect. Notch signaling components from breast cancer cells treated in culture with Resveratrol was investigated. MDA-MB-231cells were divided into control group (Res-untreated) and Res-treated groups including six concentrations 0 µM, 10 µM, 20 µM, 40 µM, 80 µM and 160 µM. Cytotoxicity test were evaluated by CCK-8. The mRNA and Protein expression levels of Notch1, Jagged1, Dll4 and Hes-5 were detected by RT-PCR and Western blot. The mRNA expression of Notch1, Jagged1, Hes-5 and Dll4 in the Res administration group decreased significantly (0.01 < p < .05), and Hes-5 and Dll4 were extremely significant (p < .01). Compared with the blank control group, the protein expression of Notch1 and Dll4 decreased significantly in each concentration Res group, but the decrease of protein expression of Jagged1 and Hes-5 was not significant. In conclusion, Res regulates mRNA and protein expression of Notch1, Dll4 of MDA-MB-231 cells via Notch pathway.

Differential Inhibition of Sox10 Functions by Notch-Hes Pathway.

In the developing central nervous system, the terminal differentiation of oligodendrocytes (OLs) is regulated by both extrinsic and intrinsic factors. Recent studies have suggested that the Notch-Hes signaling pathway influences the maturation of oligodendrocytes in culture and during development. However, the specific Notch receptors and their downstream effectors Hes genes that are involved in oligodendrocyte maturation have not been investigated systematically. In this study, we showed that Notch1 and Notch3 are expressed in oligodendrocyte precursor cells (OPCs) during gliogenesis, and Hes5 is the major Notch downstream transcription factor that is transiently expressed in OPCs. Overexpression of Notch intracellular domain (NICD) and Hes5 proteins in embryonic chicken spinal cord suppressed both the endogenous and Sox10-induced Mbp gene expression. Unexpectedly, overexpression of NICD/Hes5 did not inhibit Sox10 induction of Olig2 expression and Myrf induced Mbp expression, suggesting the differential inhibitory effects of NICD/Hes5 signaling on Sox10 activation of myelin-related genes and early progenitor genes.

DNA methylation inhibitor attenuates polyglutamine-induced neurodegeneration by regulating Hes5.

Spinal and bulbar muscular atrophy (SBMA) is a polyglutamine-mediated neuromuscular disease caused by a CAG repeat expansion in the androgen receptor (AR) gene. While transcriptional dysregulation is known to play a critical role in the pathogenesis of SBMA, the underlying molecular pathomechanisms remain unclear. DNA methylation is a fundamental epigenetic modification that silences the transcription of various genes that have a CpG-rich promoter. Here, we showed that DNA methyltransferase 1 (Dnmt1) is highly expressed in the spinal motor neurons of an SBMA mouse model and in patients with SBMA. Both genetic Dnmt1 depletion and treatment with RG108, a DNA methylation inhibitor, ameliorated the viability of SBMA model cells. Furthermore, a continuous intracerebroventricular injection of RG108 mitigated the phenotype of SBMA mice. DNA methylation array analysis identified hairy and enhancer of split 5 (Hes5) as having a CpG island with hyper-methylation in the promoter region, and the Hes5 expression was strongly silenced in SBMA. Moreover, Hes5 over-expression rescued the SBMA cells possibly by inducing Smad2 phosphorylation. Our findings suggest DNA hyper-methylation underlies the neurodegeneration in SBMA.

Exploration of the Notch3-HES5 signal pathway in monocrotaline-induced pulmonary hypertension using rat model.

OBJECTIVE: This study explores the role of the Notch3-HES5 signal pathway in monocrotaline-induced pulmonary hypertension (PH) using rat models. METHOD: Sprague Dawley rats (n = 45) were randomly grouped into normal group, control group, and model group. Rats in the model group were used to establish the PH rat model. Four weeks after model establishment, right catheterization was used to measure the mean pulmonary arterial pressure (mPAP) and right ventricular systolic pressure (RVSP) to analyze hemodynamic changes. The severity of PH was assessed by the right ventricular hypertrophy index (RVHI) and percentage of media thickness (MT%). The expressions of Notch3 and HES5 were determined by ELISA and reverse transcription-polymerase chain reaction. The correlation of mRNA expressions of Notch3 and HES5 with mPAP was analyzed. RESULTS: Rats in the model group had higher mPAP, RVSP, RVHI, and MT% as well as thicker pulmonary arterioles wall than those in the normal group. Immunohistochemistry showed Notch3 and HES5 were mainly expressed in the smooth muscle cell in pulmonary arterioles. In comparison with the normal group, rats in the model group had elevated expressions of Notch3 and HES5. The mean pulmonary arterial pressure was positively related with mRNA expressions of Notch3 and HES5. CONCLUSION: Taken together, our study demonstrates that monocrotaline-induced PH rats had high expressions of the Notch3-HES5 signal pathway in the pulmonary arterioles. The signal of the Notch3-HES5 signal pathway was positively related to the hemodynamics of the lung vasculature.

Regulation of temporal properties of neural stem cells and transition timing of neurogenesis and gliogenesis during mammalian neocortical development.

In the developing mammalian neocortex, neural stem cells (NSCs) gradually alter their characteristics as development proceeds. NSCs initially expand the progenitor pool by symmetric proliferative division and then shift to asymmetric neurogenic division to commence neurogenesis. NSCs sequentially give rise to deep layer neurons first and superficial layer neurons later through mid- to late-embryonic stages, followed by shifting to a gliogenic phase at perinatal stages. The precise mechanisms regulating developmental timing of the transition from symmetric to asymmetric division have not been fully elucidated; however, gradual elongation in cell cycle length and concomitant accumulation of determinants that promote neuronal differentiation may function as a biological clock that regulates the onset of asymmetric neurogenic division. On the other hand, epigenetic regulatory systems have been implicated in the regulation of transition timing of neurogenesis and gliogenesis; the polycomb group (PcG) complex and Hmga genes have been found to govern the developmental timing by modulating chromatin structure during neocortical development. Furthermore, we uncovered several factors and mechanisms underlying the regulation of timing of neocortical neurogenesis and gliogenesis. In this review, we discuss recent findings regarding the mechanisms that govern the temporal properties of NSCs and the precise transition timing during neocortical development.

Neuroprotective effect of Notch pathway inhibitor DAPT against focal cerebral ischemia/reperfusion 3 hours before model establishment.

As an inhibitor of the Notch signaling pathway, N-[N-(3,5-difluorohenacetyl)-l-alanyl]-S-phenylglycine tert-butyl ester (DAPT) may protect brain tissue from serious ischemic injury. This study aimed to explore neuroprotection by DAPT after cerebral ischemia/reperfusion (I/R) injury. DAPT was intraperitoneally injected 3 hours before the establishment of a focal cerebral I/R model in the right middle cerebral artery of obstructed mice. Longa scores were used to assess neurological changes of mice. Nissl staining and TdT-mediated dUTP-biotin nick-end labeling staining were used to examine neuronal damage and cell apoptosis in the right prefrontal cortex, while immunofluorescence staining was used to detect glial fibrillary acidic protein- and Notch1-positive cells. Protein expression levels of Hes1 and Hes5 were detected by western blot assay in the right prefrontal cortex. Our results demonstrated that DAPT significantly improved neurobehavioral scores and relieved neuronal morphological damage. DAPT decreased the number of glial fibrillary acidic protein- and Notch1-positive cells in the right prefrontal cortex, while also reducing the number of apoptotic cells and decreasing interleukin-6 and tumor necrosis factor-α contents, and simultaneously downregulating Hes1 and Hes5 protein expression. These findings verify that DAPT alleviates pathological lesions and strengthens the anti-inflammatory response after cerebral I/R injury. Thus, DAPT might be developed as an effective drug for the prevention of cerebral I/R injury.