n-(n-(3-5-difluorophenacetyl)alanyl)phenylglycine-tert-butyl-ester and Disease-Models--Animal

Nonalcoholic fatty liver disease (NAFLD) has become the most common cause of chronic liver disease worldwide and an urgent target for clinical intervention. Notch1 signaling pathway activity was found to be related to the severity of NAFLD, but the specific mechanism is not precise. Here, we investigated the potential mechanisms of Notch1 signaling in the development of NAFLD. Firstly, we found that Notch1 signaling is activated in free fatty acids-treated HepG2 cells accompanied by lipid accumulation, apoptosis, oxidative stress, and mitochondrial damage, which could be alleviated by Notch1 inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). In the meantime, we found that administration of DAPT activated the autophagy pathway in NAFLD. Furthermore, the use of autophagy inhibitor chloroquine reversed the DAPT-mediated protective effect in NAFLD. All our results uncover a vital role of Notch1 in hepatocyte injury and metabolism of NAFLD, giving rise to a new sight for NAFLD treatment by regulation of Notch signaling and autophagy pathway.

Topics: Animals; Apoptosis; Autophagy; Cells, Cultured; Dipeptides; Disease Models, Animal; Hep G2 Cells; Hepatocytes; Humans; Male; Mice; Mice, Inbred C57BL; Non-alcoholic Fatty Liver Disease; Oxidative Stress; Receptor, Notch1; Signal Transduction

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Diabetes mellitus can exacerbate renal ischemia‑reperfusion (I/R) injury (RI/RI) in diabetic rats. Previous studies have shown that Notch signaling is involved in renal disorders. The aim of the present study was to evaluate the protective effect of the Notch inhibitor γ‑secretase N‑[N‑(3,5‑difluorophenacetyl)‑L‑alanyl]‑S‑phenylglycine t‑butyl ester (DAPT) on RI/RI in a streptozocin (STZ)‑induced diabetic rat model. STZ‑induced diabetic rats were randomly grouped for different treatments. Cisplatin was used to trigger the Notch signaling pathway and the animals were preconditioned with DAPT to block the signaling pathway. Renal function, oxidative stress and inflammatory factors were examined. DAPT‑treated diabetic rats demonstrated mitigated renal injury and function, antioxidative activity was significantly improved and HIF‑1a was upregulated. Notch inhibitor DAPT is a potential therapeutic target to improve the outcome of RI/RI in STZ‑induced diabetic rats in part via the regulation of anti‑oxidation and HIF‑1a.

Topics: Animals; Blood Urea Nitrogen; Cisplatin; Creatinine; Diabetes Mellitus, Experimental; Dipeptides; Disease Models, Animal; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation Mediators; Kidney; Male; Oxidation-Reduction; Rats, Sprague-Dawley; Receptors, Notch; Reperfusion Injury; RNA, Messenger; Signal Transduction; Streptozocin

Understanding the signaling pathways that regulate proliferation and differentiation of muscle progenitors is essential for successful cell transplantation for treatment of Duchenne muscular dystrophy. Here, we report that a γ-secretase inhibitor, DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine tertial butyl ester), which inhibits the release of NICD (Notch intercellular domain), promotes the fusion of human muscle progenitors in vitro and improves their engraftment in the tibialis anterior muscle of immune-deficient mice. Gene expression analysis revealed that DAPT severely down-regulates PTGER2, which encodes prostaglandin (PG) E2 receptor 2 (EP2), in human muscle progenitors in the differentiation condition. Functional analysis suggested that Notch signaling inhibits differentiation and promotes self-renewal of human muscle progenitors via PGE2/EP2 signaling in a cAMP/PKA-independent manner.

Topics: Amyloid Precursor Protein Secretases; Animals; Cell Differentiation; Cell Fusion; Cell Line; Cell Proliferation; Cell Self Renewal; Cell Survival; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dinoprostone; Dipeptides; Disease Models, Animal; Enzyme Inhibitors; Humans; Male; Mice, Inbred mdx; Mice, Inbred NOD; Mice, SCID; Muscle Development; Muscle Fibers, Skeletal; Muscle, Skeletal; Myoblasts, Skeletal; Receptor, Notch3; Receptors, Prostaglandin E, EP2 Subtype; Signal Transduction; Stem Cell Transplantation

CHARGE syndrome is an autosomal dominant congenital disorder caused primarily by mutations in the CHD7 gene. Using a small molecule screen in a zebrafish model of CHARGE syndrome, we identified 4 compounds that rescue embryos from disease-like phenotypes. Our screen yielded DAPT, a Notch signaling inhibitor that could ameliorate the craniofacial, cranial neuronal and myelination defects in chd7 morphant zebrafish embryos. We discovered that Procainamide, an inhibitor of DNA methyltransferase 1, was able to recover the pattern of expression of isl2a, a cranial neuronal marker while also reducing the effect on craniofacial cartilage and myelination. M344, an inhibitor of Histone deacetylases had a strong recovery effect on craniofacial cartilage defects and could also modestly revert the myelination defects in zebrafish embryos. CHIC-35, a SIRT1 inhibitor partially restored the expression of isl2a in cranial neurons while causing a partial reversion of myelination and craniofacial cartilage defects. Our results suggest that a modular approach to phenotypic rescue in multi-organ syndromes might be a more successful approach to treat these disorders. Our findings also open up the possibility of using these compounds for other disorders with shared phenotypes.

Topics: Animals; Animals, Genetically Modified; Cartilage; CHARGE Syndrome; Dipeptides; Disease Models, Animal; DNA (Cytosine-5-)-Methyltransferase 1; DNA Helicases; DNA-Binding Proteins; Embryo, Nonmammalian; Gene Knockdown Techniques; Histone Deacetylase Inhibitors; LIM-Homeodomain Proteins; Nerve Fibers, Myelinated; Neurons; Procainamide; Receptors, Notch; Sirtuin 1; Transcription Factors; Vorinostat; Zebrafish; Zebrafish Proteins

Abdominal aortic aneurysm (AAA) is characterized by transmural infiltration of myeloid cells at the vascular injury site. Previously, we reported preventive effects of Notch deficiency on the development of AAA by reduction of infiltrating myeloid cells. In this study, we examined if Notch inhibition attenuates the progression of pre-established AAA and potential implications. Pharmacological Notch inhibitor (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester; DAPT) was administered subcutaneously three times a week starting at day 28 of angiotensin II (AngII) infusion. Progressive increase in pulse wave velocity (PWV), maximal intra-luminal diameter (MILD) and maximal external aortic diameter (MEAD) were observed at day 56 of the AngII. DAPT prevented such increase in MILD, PWV and MEAD (P < 0.01). Histologically, the aortae of DAPT-treated Apoe

Topics: ADP-ribosyl Cyclase 1; Angiotensin II; Animals; Aorta; Aortic Aneurysm, Abdominal; Cells, Cultured; Collagen; Cytokines; Dipeptides; Disease Models, Animal; Extracellular Matrix; Gene Expression Regulation; Humans; Male; Membrane Glycoproteins; Mice; Myocytes, Smooth Muscle; Receptors, Notch; Signal Transduction

BACKGROUND Retinopathy of prematurity (ROP), or retrolental fibroplasia, affects premature infants who have undergone intensive care with oxygen therapy. This study aimed to investigate the inhibitory effect of the gamma-secretase inhibitor, DAPT, on neovascularization and its mechanism in a rat model of ROP. MATERIAL AND METHODS Sixty neonatal Sprague-Dawley (SD) rats included the control group (n=20), the model group (n=20), and the DAPT-treated group (n=20). The rat model of ROP was established using repeat cycles of oxygen inhalation. Enzyme-linked immunosorbent assay (ELISA) measured serum levels of vascular endothelial growth factor (VEGF), VEGF receptor-1 (VEGFR-1), and VEGFR-2. Histology of the retinal tissue included immunohistochemistry for the expression of Notch homolog-1 (Notch-1) and delta-like ligand 4 (DLL4). Retinal mRNA levels of DLL4, Notch-1, VEGF, VEGFR-1, and VEGFR-2 were evaluated with quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS The rat model of ROP showed increased serum levels of VEGF, VEGFR-1, and VEGFR-2 compared with the control group, which were decreased in the DAPT group. Histology of the retinal tissue in the model group showed degeneration of the retinal ganglion cells, and immunohistochemistry showed increased expression of Notch-1 and DLL4 compared with the control group and DAPT group. Retinal tissue in the model group had increased mRNA levels of DLL4, Notch-1, VEGF, VEGFR-1, and VEGFR-2 compared with the control group, and the DAPT group. CONCLUSIONS In a rat model, treatment with DAPT reduced the retinal changes associated with ROP with a mechanism that involved VEGF and its receptors through the DLL4/Notch-1 pathway.

Topics: Amyloid Precursor Protein Secretases; Animals; Animals, Newborn; Dipeptides; Disease Models, Animal; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Neovascularization, Pathologic; Rats; Rats, Sprague-Dawley; Receptor, Notch1; Retinopathy of Prematurity; Signal Transduction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2

Autism spectrum disorders (ASDs) comprise a number of heterogeneous neurodevelopmental diseases. Recent studies suggest that the abnormal transmission of neural signaling pathways is associated with the pathogenesis of autism. The aim of this study was to identify a link between the Notch signaling pathway and the pathogenesis of autism. In this study, we demonstrated that prenatal exposure to valproic acid (VPA) resulted in autistic-like behaviors in offspring rats and that the expression of the Notch signaling pathway-related molecules Notch1, Jagged1, Notch intracellular domain (NICD) and Hes1 increased in the prefrontal cortex (PFC), hippocampus (HC) and cerebellum (CB) of VPA rats compared to those of controls. However, inhibiting the Notch pathway with (3,5-Difluorophenacetyl)-L-alanyl-S-phenylglycine-2-butyl Ester (Dapt) reduced the overexpression of Notch pathway-related molecules in offspring rats. Notably, Dapt improved autistic-like behaviors in a VPA-exposed rat model of autism. Furthermore, we investigated whether Dapt improved autistic-like behavior in a VPA rat model by regulating autophagy and affecting the morphology of dendritic spines. We found that the expression of the autophagy-related proteins Beclin 1, LC3B and phospho-p62 in the PFC, HC and CB of VPA model rats increased after Notch signal activation and was inhibited by Dapt compared to those of controls. Moreover, postsynaptic density-95 (PSD-95) protein expression also increased significantly compared to that of VPA model rats. The density of dendritic spines decreased in the PFC of VPA rats treated with Dapt compared to that of VPA model rats. Our present results suggest that VPA induces an abnormal activation of the Notch signaling pathway. The inhibition of excessive Notch signaling activation by Dapt can alleviate autistic-like behaviors in VPA rats. Our working model suggests that the Notch signaling pathway participates in the pathogenesis of autism by regulating autophagy and affecting dendritic spine growth. The results of this study may help to elucidate the mechanism underlying autism and provide a potential strategy for treating autism.

Topics: Animals; Atrophy; Autistic Disorder; Autophagy; Beclin-1; Behavior, Animal; Cerebellum; Dendritic Spines; Dipeptides; Disease Models, Animal; Disks Large Homolog 4 Protein; Female; Hippocampus; Male; Microtubule-Associated Proteins; Phosphorylation; Prefrontal Cortex; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Receptor, Notch1; Sequestosome-1 Protein; Signal Transduction; Valproic Acid

OBJECTIVE Reactive astrogliosis, a key feature that is characterized by glial proliferation, has been observed in rat brains after intracerebral hemorrhage (ICH). However, the mechanisms that control reactive astrogliosis formation remain unknown. Notch-1 signaling plays a critical role in modulating reactive astrogliosis. The purpose of this paper was to establish whether Notch-1 signaling is involved in reactive astrogliosis after ICH. METHODS ICH was induced in adult male Sprague-Dawley rats via stereotactic injection of autologous blood into the right globus pallidus. N-[ N-(3,5-difluorophenacetyl)-l-alanyl]- S-phenylglycine t-butyl ester (DAPT) was injected into the lateral ventricle to block Notch-1 signaling. The rats' brains were perfused to identify proliferating cell nuclear antigen (PCNA)-positive/GFAP-positive nuclei. The expression of GFAP, Notch-1, and the activated form of Notch-1 (Notch intracellular domain [NICD]) and its ligand Jagged-1 was assessed using immunohistochemical and Western blot analyses, respectively. RESULTS Notch-1 signaling was upregulated and activated after ICH as confirmed by an increase in the expression of Notch-1 and NICD and its ligand Jagged-1. Remarkably, blockade of Notch-1 signaling with the specific inhibitor DAPT suppressed astrocytic proliferation and GFAP levels caused by ICH. In addition, DAPT improved neurological outcome after ICH. CONCLUSIONS Notch-1 signaling is a critical regulator of ICH-induced reactive astrogliosis, and its blockage may be a potential therapeutic strategy for hemorrhagic injury.

Topics: Animals; Astrocytes; Brain; Cell Proliferation; Cerebral Hemorrhage; Dipeptides; Disease Models, Animal; Glial Fibrillary Acidic Protein; Gliosis; Jagged-1 Protein; Male; Rats; Rats, Sprague-Dawley; Receptor, Notch1; Signal Transduction; Up-Regulation

Endothelial upregulation of adhesion molecules serves to recruit leukocytes to inflammatory sites and appears to be promoted by NOTCH1; however, current models based on interactions between active NOTCH1 and NF-κB components cannot explain the transcriptional selectivity exerted by NOTCH1 in this context.. Observing that Cre/Lox-induced conditional mutations of endothelial Notch modulated inflammation in murine contact hypersensitivity, we found that IL (interleukin)-1β stimulation induced rapid recruitment of RELA (v-rel avian reticuloendotheliosis viral oncogene homolog A) to genomic sites occupied by NOTCH1-RBPJ (recombination signal-binding protein for immunoglobulin kappa J region) and that NOTCH1 knockdown reduced histone H3K27 acetylation at a subset of NF-κB-directed inflammatory enhancers.. Our findings reveal that NOTCH1 signaling supports the expression of a subset of inflammatory genes at the enhancer level and demonstrate how key signaling pathways converge on chromatin to coordinate the transition to an infla mmatory endothelial phenotype.

Topics: Acetylation; Animals; Appendicitis; Cells, Cultured; Dermatitis, Contact; Dipeptides; Disease Models, Animal; Endothelial Cells; Female; Gene Expression Regulation; Histones; Human Umbilical Vein Endothelial Cells; Humans; Immunoglobulin J Recombination Signal Sequence-Binding Protein; Inflammation; Interleukin-1beta; Male; Mice, Inbred C57BL; Mice, Transgenic; Phenotype; Receptor, Notch1; Signal Transduction; Transcription Factor RelA

The Notch signaling pathway is involved in angiogenesis induced by brain ischemia and can be efficiently inhibited by the. Sprague-Dawley rats (. SWI showed that DAPT treatment significantly enhanced angiogenesis in the ischemic boundary zone (IBZ) with respect to the control group, with local CBF in the angiogenic area elevated, along with increases in vascular density confirmed by histology.. Treatment of ischemic stroke with DAPT significantly augments angiogenesis, which promotes poststroke brain remodeling by elevating CBF level, and these processes can be dynamically monitored and evaluated by MRI.

Topics: Animals; Brain Ischemia; Cerebrovascular Circulation; Dipeptides; Disease Models, Animal; Magnetic Resonance Imaging; Male; Neovascularization, Physiologic; Rats; Rats, Sprague-Dawley; Receptors, Notch; Signal Transduction; Time Factors

Inflammation-dominated sympathetic sprouting adjacent to the necrotic region following myocardial infarction (MI) has been implicated in the etiology of arrhythmias resulting in sudden cardiac death; however, the mechanisms responsible remain to be elucidated. Although being a key immune mediator, the role of Notch has yet to be explored. We investigated whether Notch regulates macrophage responses to inflammation and affects cardiac sympathetic reinnervation in rats undergoing MI. MI was induced by coronary artery ligation. A high level of Notch intracellular domain was observed in the macrophages that infiltrated the infarct area at 3 days post-MI. The administration of the Notch inhibitor N-N-(3,5-difluorophenacetyl-L-alanyl)-S-phenylglycine-t-butyl ester (DAPT) (intravenously 30 min before MI and then daily until death) decreased the number of macrophages and significantly increased the M2 macrophage activation profile in the early stages and attenuated the expression of nerve growth factor (NGF). Eventually, NGF-induced sympathetic hyperinnervation was blunted, as assessed by the immunofluorescence of tyrosine hydroxylase. At 7 days post-MI, the arrhythmia score of programmed electric stimulation in the vehicle-treated infarcted rats was higher than that in rats treated with DAPT. Further deterioration in cardiac function and decreases in the plasma levels of TNF-α and IL-1β were also detected. In vitro studies revealed that LPS/IFN-γ upregulated the surface expression of NGF in M1 macrophages in a Notch-dependent manner. We concluded that Notch inhibition during the acute inflammatory response phase is associated with the downregulation of NGF, probably through a macrophage-dependent pathway, thus preventing the process of sympathetic hyperinnervation.

Topics: Animals; Anti-Inflammatory Agents; Arrhythmias, Cardiac; Cardiac Pacing, Artificial; Cells, Cultured; Dipeptides; Disease Models, Animal; Heart; Inflammation Mediators; Interleukin-1beta; Macrophages; Male; Myocardial Infarction; Myocardium; Nerve Growth Factor; Phenotype; Rats, Sprague-Dawley; Receptor, Notch1; Signal Transduction; Sympathetic Nervous System; Tumor Necrosis Factor-alpha

To test the hypothesis that Notch signalling plays a role in the pathogenesis of rheumatoid arthritis (RA) and to determine whether pharmacological inhibition of Notch signalling with γ-secretase inhibitors can ameliorate the RA disease process in an animal model.. Collagen-induced arthritis was induced in C57BL/6 or Notch antisense transgenic mice by immunisation with chicken type II collagen (CII). C57BL/6 mice were administered with different doses of inhibitors of γ-secretase, an enzyme required for Notch activation, at disease onset or after onset of symptoms. Severity of arthritis was monitored by clinical and histological scores, and in vivo non-invasive near-infrared fluorescence (NIRF) images. Micro-CT was used to confirm joint destruction. The levels of CII antibodies and cytokines in serum were determined by ELISA and bead-based cytokine assay. The expression levels of cytokines were studied by quantitative PCR in rheumatoid synovial fibroblasts.. The data show that Notch signalling stimulates synoviocytes and accelerates their production of proinflammatory cytokines and immune responses involving the upregulation of IgG1 and IgG2a. Pharmacological inhibition of γ-secretase and antisense-mediated knockdown of Notch attenuates the severity of inflammatory arthritis, including arthritis indices, paw thickness, tissue damage and neutrophil infiltration, and reduces the levels of active NF-κB, ICAM-1, proinflammatory cytokines and matrix metalloproteinase-3 activity in the mouse model of RA.. These results suggest that Notch is involved in the pathogenesis of RA and that inhibition of Notch signalling is a novel approach for treating RA.

Topics: Amyloid Precursor Protein Secretases; Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Cytokines; Dipeptides; Disease Models, Animal; Mice; Mice, Inbred C57BL; Mice, Transgenic; Receptors, Notch; Severity of Illness Index; Signal Transduction; Synovial Membrane

Relaxin (RLX) can prevent cardiac fibrosis. We aimed to investigate the possible mechanism and signal transduction pathway of RLX inhibiting cardiac fibrosis.. Isoproterenol (5 mg·kg(-1)·d(-1)) was used to establish the cardiac fibrosis model in rats, which were administered RLX. The cardiac function, related targets of cardiac fibrosis, and endothelial-mesenchymal transition (EndMT) were measured. Transforming growth factor β (TGF-β) was used to induce EndMT in human umbilical vein endothelial cells, which were pretreated with RLX, 200 ng·mL(-1), then with the inhibitor of Notch. Transwell cell migration was used to evaluate cell migration. CD31 and vimentin content was determined by immunofluorescence staining and Western blot analysis. Notch protein level was examined by Western blot analysis.. RLX improved cardiac function in rats with cardiac fibrosis; it reduced the content of collagen I and III, increased the microvascular density of the myocardium, and suppressed the EndMT in heart tissue. In vitro, RLX decreased the mobility of human umbilical vein endothelial cells induced by TGF-β, increased the expression of endothelial CD31, and decreased vimentin content. Compared to TGF-β and RLX co-culture alone, TGF-β + RLX + Notch inhibitor increased cell mobility and the EndMT, but decreased the levels of Notch-1, HES-1, and Jagged-1 proteins.. RLX may inhibit the cardiac fibrosis via EndMT by Notch-mediated signaling.

Topics: Animals; Cardiomyopathies; Cell Movement; Cells, Cultured; Collagen; Dipeptides; Disease Models, Animal; Endothelial Cells; Epithelial-Mesenchymal Transition; Fibrosis; Human Umbilical Vein Endothelial Cells; Humans; Isoproterenol; Male; Myocardium; Neovascularization, Physiologic; Rats, Sprague-Dawley; Receptor, Notch1; Relaxin; Signal Transduction; Ventricular Function, Left

Alzheimer's disease (AD) is a neurodegenerative disorder which mainly affects elderly population. MicroRNAs (miRNA) are small RNA molecules that fine-tune gene expression at posttranscriptional level and exert important functions in AD. MicroRNA-124 (miR-124) is a kind of miRNA abundantly expressed in the central nervous system. It is highly conserved from Caenorhabditis elegans to humans. However, its function in AD is still elusive. In this study, we found miR-124 was significantly down-regulated in AD flies. miR-124 mutant flies showed impaired climbing ability and shortened lifespan. In contrast, miR-124 expression rescued locomotive defects of AD flies. Using microarray analysis to test gene expression profiles of miR-124 mutant flies, we found that Notch signaling pathway was potentially targeted by miR-124. Further experiments showed that miR-124 regulated Notch ligand Delta expression by acting on specific site of Delta 3`UTR. In addition, reduced Delta expression by RNA interference extended lifespan and ameliorated learning defects of AD Drosophila. Notch inhibitor DAPT could also alleviate AD phenotypes, which confirmed our findings. In conclusion, our study indicates that miR-124 plays neuroprotective roles in AD Drosophila by targeting Delta in Notch signaling pathway, which helps further our understanding of miRNAs in the molecular pathology of AD.

Topics: 3' Untranslated Regions; Alzheimer Disease; Animals; Binding Sites; Conditioning, Operant; Dipeptides; Disease Models, Animal; Drosophila melanogaster; Female; Gene Expression Profiling; Gene Expression Regulation; Humans; Intracellular Signaling Peptides and Proteins; Locomotion; Longevity; Male; Membrane Proteins; Microarray Analysis; MicroRNAs; Protein Binding; RNA, Small Interfering; Signal Transduction

Perioperative discontinuation of aspirin is often considered due to bleeding concern. We determined whether this discontinuation affected neurological outcome after brain ischemia.. Adult male Sprague-Dawley rats were subjected to a 90-minute right middle cerebral arterial occlusion (MCAO). They received 30 mg/kg/day aspirin via gastric gavage: 1) for 2 days at 5 days before MCAO; 2) for 2 days at 5 days before MCAO and for 3 days after MCAO; 3) for 7 days before MCAO; or 4) for 7 days before MCAO and for 3 days after MCAO. Neurological outcome was evaluated 3 days after the MCAO. Ischemic penumbral cortex was harvested 1 or 3 days after MCAO for determining Notch intracellular domain (NICD), IL-6 and IL-1β levels.. Aspirin given by regimen 2 and 3 but not by regimen 1 improved neurological outcome. Neuroprotection was achieved by N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a Notch activation inhibitor. DAPT and aspirin given only by regimen 2 and 3 reduced NICD, IL-6 and IL-1β in the ischemic penumbral cortex. NICD was found in microglial nuclei. Microglial activation in the ischemic tissues was inhibited by aspirin.. Aspirin use during the perioperative period provides neuroprotection. Inhibition of Notch activation and neuroinflammation may contribute to the neuroprotection of aspirin.

Topics: Analysis of Variance; Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspirin; Brain Infarction; Dipeptides; Disease Models, Animal; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Infarction, Middle Cerebral Artery; Interleukin-1beta; Interleukin-6; Male; Nervous System Diseases; Psychomotor Performance; Rats, Sprague-Dawley; Time Factors

Notch signaling deregulation is linked to the onset of several tumors including T-cell acute lymphoblastic leukemia (T-ALL). Deregulated microRNA (miRNA) expression is also associated with several cancers, including leukemias. However, the transcriptional regulators of miRNAs, as well as the relationships between Notch signaling and miRNA deregulation, are poorly understood. To identify miRNAs regulated by Notch pathway, we performed microarray-based miRNA profiling of several Notch-expressing T-ALL models. Among seven miRNAs, consistently regulated by overexpressing or silencing Notch3, we focused our attention on miR-223, whose putative promoter analysis revealed a conserved RBPjk binding site, which was nested to an NF-kB consensus. Luciferase and chromatin immunoprecipitation assays on the promoter region of miR-223 show that both Notch and NF-kB are novel coregulatory signals of miR-223 expression, being able to activate cooperatively the transcriptional activity of miR-223 promoter. Notably, the Notch-mediated activation of miR-223 represses the tumor suppressor FBXW7 in T-ALL cell lines. Moreover, we observed the inverse correlation of miR-223 and FBXW7 expression in a panel of T-ALL patient-derived xenografts. Finally, we show that miR-223 inhibition prevents T-ALL resistance to γ-secretase inhibitor (GSI) treatment, suggesting that miR-223 could be involved in GSI sensitivity and its inhibition may be exploited in target therapy protocols.

Topics: Amyloid Precursor Protein Secretases; Animals; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cluster Analysis; Dipeptides; Disease Models, Animal; Drug Resistance, Neoplasm; F-Box Proteins; F-Box-WD Repeat-Containing Protein 7; Gene Expression Profiling; Gene Expression Regulation, Leukemic; Gene Silencing; Humans; Mice, Transgenic; MicroRNAs; NF-kappa B; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Receptors, Notch; RNA Interference; Signal Transduction; Ubiquitin-Protein Ligases

Recently we have shown that the highly conserved herpes simplex virus glycoprotein K (gK) binds to signal peptide peptidase (SPP), also known as minor histocompatibility antigen H13. In this study we have demonstrated for the first time that inhibitors of SPP, such as L685,458, (Z-LL)2 ketone, aspirin, ibuprofen and DAPT, significantly reduced HSV-1 replication in tissue culture. Inhibition of SPP activity via (Z-LL)2 ketone significantly reduced viral transcripts in the nucleus of infected cells. Finally, when administered during primary infection, (Z-LL)2 ketone inhibitor reduced HSV-1 replication in the eyes of ocularly infected mice. Thus, blocking SPP activity may represent a clinically effective and expedient approach to the reduction of viral replication and the resulting pathology.

Topics: Animals; Aspartic Acid Endopeptidases; Aspirin; Carbamates; Cell Fractionation; Cells, Cultured; Dipeptides; Disease Models, Animal; DNA, Viral; Enzyme Inhibitors; Female; Gene Expression Regulation, Viral; Herpesvirus 1, Human; Ibuprofen; Keratitis, Herpetic; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Ophthalmic Solutions; Rabbits; Real-Time Polymerase Chain Reaction; RNA, Messenger; Skin; Virus Replication

NOTCH-dependent signaling pathways are critical for normal bone remodeling; however, it is unclear if dysfunctional NOTCH activation contributes to inflammation-mediated bone loss, as observed in rheumatoid arthritis (RA) patients. We performed RNA sequencing and pathway analyses in mesenchymal stem cells (MSCs) isolated from transgenic TNF-expressing mice, a model of RA, to identify pathways responsible for decreased osteoblast differentiation. 53 pathways were dysregulated in MSCs from RA mice, among which expression of genes encoding NOTCH pathway members and members of the noncanonical NF-κB pathway were markedly elevated. Administration of NOTCH inhibitors to RA mice prevented bone loss and osteoblast inhibition, and CFU-fibroblasts from RA mice treated with NOTCH inhibitors formed more new bone in recipient mice with tibial defects. Overexpression of the noncanonical NF-κB subunit p52 and RELB in a murine pluripotent stem cell line increased NOTCH intracellular domain-dependent (NICD-dependent) activation of an RBPjκ reporter and levels of the transcription factor HES1. TNF promoted p52/RELB binding to NICD, which enhanced binding at the RBPjκ site within the Hes1 promoter. Furthermore, MSC-enriched cells from RA patients exhibited elevated levels of HES1, p52, and RELB. Together, these data indicate that persistent NOTCH activation in MSCs contributes to decreased osteoblast differentiation associated with RA and suggest that NOTCH inhibitors could prevent inflammation-mediated bone loss.

Topics: Animals; Arthritis, Rheumatoid; Basic Helix-Loop-Helix Transcription Factors; Bone Resorption; Cell Differentiation; Dipeptides; Disease Models, Animal; Gene Expression; Homeodomain Proteins; Humans; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; NF-kappa B; NF-kappa B p52 Subunit; Osteoblasts; Promoter Regions, Genetic; Receptors, Notch; Signal Transduction; Transcription Factor HES-1; Transcription Factor RelB; Tumor Necrosis Factor-alpha

The progression of abdominal aortic aneurysm (AAA) involves a sustained influx of proinflammatory macrophages, which exacerbate tissue injury by releasing cytokines, chemokines, and matrix metalloproteinases. Previously, we showed that Notch deficiency reduces the development of AAA in the angiotensin II-induced mouse model by preventing infiltration of macrophages. Here, we examined whether Notch inhibition in this mouse model prevents progression of small AAA and whether these effects are associated with altered macrophage differentiation.. Treatment with pharmacological Notch inhibitor (DAPT [N-(N-[3,5-difluorophenacetyl]-L-alanyl)-S-phenylglycine t-butyl ester]) at day 3 or 8 of angiotensin II infusion arrested the progression of AAA in Apoe(-/-) mice, as demonstrated by a decreased luminal diameter and aortic width. The abdominal aortas of Apoe(-/-) mice treated with DAPT showed decreased expression of matrix metalloproteinases and presence of elastin precursors including tropoelastin and hyaluronic acid. Marginal adventitial thickening observed in the aorta of DAPT-treated Apoe(-/-) mice was not associated with increased macrophage content, as observed in the mice treated with angiotensin II alone. Instead, DAPT-treated abdominal aortas showed increased expression of Cd206-positive M2 macrophages and decreased expression of Il12-positive M1 macrophages. Notch1 deficiency promoted M2 differentiation of macrophages by upregulating transforming growth factor β2 in bone marrow-derived macrophages at basal levels and in response to IL4. Protein expression of transforming growth factor β2 and its downstream effector pSmad2 also increased in DAPT-treated Apoe(-/-) mice, indicating a potential link between Notch and transforming growth factor β2 signaling in the M2 differentiation of macrophages.. Pharmacological inhibitor of Notch signaling prevents the progression of AAA by macrophage differentiation-dependent mechanisms. The study also provides insights for novel therapeutic strategies to prevent the progression of small AAA.

Topics: Aged; Angiotensin II; Animals; Aorta, Abdominal; Aortic Aneurysm, Abdominal; Apolipoproteins E; Cell Differentiation; Cells, Cultured; Collagen; Dipeptides; Disease Models, Animal; Disease Progression; Elastin; Gene Expression Regulation; Humans; Macrophages; Male; Matrix Metalloproteinases; Mice, Knockout; Middle Aged; Receptor, Notch1; Signal Transduction; Time Factors; Transforming Growth Factor beta2

In the present study, we determined whether Phosphoinositide 3-kinase (PI3K) and Notch signal pathways are involved in the expression of cyclinD1, cyclinA and p27kip1 which were key molecules in controlling cell cycling from CD4(+) T lymphocyte in animal model of asthma.. Ovalbumin (OVA) sensitized murine model of asthma was used to investigate the expression of cyclin D1, cyclin A, and p27kip1 by splenic CD4(+) T lymphocytes. We further observed the effect of specific inhibitor of PI3K(LY294002) and specific inhibitor of Notch(DAPT) on the proliferation of such CD4(+) T lymphocytes.. We found that the expression of cyclinD1 and cyclinA was upregulated at both protein and mRNA levels in asthma group while p27kip1 was down-regulated. Both LY294002 and DAPT inhibit the proliferation of CD4(+) T lymphocytes in a time- and dose-dependent manner. Furthermore, LY294002 and DAPT have additive effect in down-regulation of cyclinD1 and upregulation of p27kip1. An upregulation of cyclinA, although not statistically significant, was also observed.. These data suggested that PI3K signal pathway and Notch signal pathway may coordinately regulate the cell proliferation and differentiation processes through up-regulating cyclinD1 and down-regulating p27kip1 of CD4(+) T lymphocytes.

Topics: Animals; Asthma; CD4-Positive T-Lymphocytes; Cell Proliferation; Chromones; Cyclin A; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Dipeptides; Disease Models, Animal; Dose-Response Relationship, Drug; Down-Regulation; Male; Mice; Mice, Inbred BALB C; Morpholines; Ovalbumin; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Receptors, Notch; RNA, Messenger; Signal Transduction; Spleen; Time Factors; Up-Regulation

Notch signaling plays a critical role in the maintenance of intestinal crypt epithelial cell proliferation. The aim of this study was to investigate the role of Notch signaling in the proliferation and regeneration of intestinal epithelium after intestinal ischemia reperfusion (I/R) injury.. Male Sprague-Dawley rats were subjected to sham operation or I/R by occlusion of the superior mesenteric artery (SMA) for 20 min. Intestinal tissue samples were collected at 0, 1, 2, 4, and 6 h after reperfusion. Proliferation of the intestinal epithelium was evaluated by immunohistochemical staining of proliferating nuclear antigen (PCNA). The mRNA and protein expression levels of Notch signaling components were examined using Real-time PCR and Western blot analyses. Immunofluorescence was also performed to detect the expression and location of Jagged-2, cleaved Notch-1, and Hes-1 in the intestine. Finally, the γ-secretase inhibitor DAPT and the siRNA for Jagged-2 and Hes-1 were applied to investigate the functional role of Notch signaling in the proliferation of intestinal epithelial cells in an in vitro IEC-6 culture system.. I/R injury caused increased intestinal crypt epithelial cell proliferation and increased mRNA and protein expression of Jagged-2, Notch-1, and Hes-1. The immunofluorescence results further confirmed increased protein expression of Jagged-2, cleaved Notch-1, and Hes-1 in the intestinal crypts. The inhibition of Notch signaling with DAPT and the suppression of Jagged-2 and Hes-1 expression using siRNA both significantly inhibited the proliferation of IEC-6 cells.. The Jagged-2/Notch-1/Hes-1 signaling pathway is involved in intestinal epithelium regeneration early after I/R injury by increasing crypt epithelial cell proliferation.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Line; Cell Proliferation; Dipeptides; Disease Models, Animal; Gene Silencing; Homeodomain Proteins; Intestinal Mucosa; Jagged-2 Protein; Male; Membrane Proteins; Proliferating Cell Nuclear Antigen; Rats; Receptor, Notch1; Regeneration; Reperfusion Injury; RNA Interference; Signal Transduction; Transcription Factor HES-1

Cholangiocacinoma (CC) is a cancer disease with rising incidence. Notch signaling has been shown to be deregulated in many cancers. However, the role of this signaling pathway in the carcinogenesis of CC is still not fully explored. In this study, we investigated the effects of Notch inhibition by γ-secretase inhibitor IX (GSI IX) in cultured human CC cell lines and we established a transgenic mouse model with liver specific expression of the intracellular domain of Notch (Notch-ICD) and inactivation of tumor suppressor p53. GSI IX treatment effectively impaired cell proliferation, migration, invasion, epithelial to mesenchymal transition and growth of softagar colonies. In vivo overexpression of Notch-ICD together with an inactivation of p53 significantly increased tumor burden and showed CC characteristics.. Our study highlights the importance of Notch signaling in the tumorigenesis of CC and demonstrates that additional inactivation of p53 in vivo.

Topics: Amyloid Precursor Protein Secretases; Animals; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cholangiocarcinoma; Dipeptides; Disease Models, Animal; Enzyme Inhibitors; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Infant; Male; Mice; Neoplasm Invasiveness; Protein Structure, Tertiary; Receptor, Notch1; Signal Transduction; Tumor Suppressor Protein p53

The objective of the current study was to investigate the regulation of VEGF signaling and tumor angiogenesis by gamma-secretase inhibitor DAPT in glioblastoma. Effects of DAPT on VEGFR1, VEGFR2, endothelial cell proliferation and vessel function were evaluated using mouse microvascular endothelial H5V cell line and U87MG xenograft mouse models. We found that DAPT efficiently inhibited Notch signaling, increased VEGFR2 expression, but decreased VEGFR1 expression. DAPT treatment enhanced endothelial cell proliferation when used combined with VEGF, but exerted no effect if used alone. In U87MG xenograft mouse models, DAPT treatment increased tumor vessel density but compromised vessel function, as evidenced by poor perfusion and aggravated hypoxia. Therefore, DAPT treatment results in an uncoupling of tumor vessel density from vessel function and suppresses glioblastoma growth; disturbance of angiogenesis with DAPT presents a novel therapeutic approach for glioblastoma.

Topics: Amyloid Precursor Protein Secretases; Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Dipeptides; Disease Models, Animal; Endothelial Cells; Female; Glioblastoma; Mice; Mice, Inbred BALB C; Neovascularization, Pathologic; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2

Tight junction protein degradation is a principal characteristic of the blood-brain barrier (BBB) damage that occurs during brain ischemia.. We investigated the mechanisms of occludin degradation that underlie permanent middle cerebral artery occlusion (pMCAO) in rats.. Western blot and Co-immunoprecipitation data indicated ubiquitination and degradation of occludin in brain after pMCAO, which was consistent with ZO-1 degradation in penumbra regions as observed at 24 h after pMCAO. We further investigated candidate protease(s) responsible for the degradation of occludin during pMCAO. The intraventricular administration of γ-secretase blocker DAPT significantly inhibited the pMCAO-induced neurovascular damage, whereas ALLM and Batimastat, which are inhibitors of calpain and metalloproteinase proteases, respectively, were less effective. Notably, we found that DAPT significantly inhibited BBB disruption in comparison with vehicle treatment, as assessed by Evans blue excretion. Interestingly, the confocal immunostaining revealed that activation of the E3 ubiquitin ligase Itch is associated with degradation of occludin in brain microvessels following ischemia. Furthermore, our data demonstrate that the inhibition of γ-secretase signaling and the itch-mediated ubiquitination of occludin likely underlie the vasoprotective effect of DAPT after pMCAO.. The γ-secretase blocker DAPT reduces the permeability of the BBB by decreasing the ubiquitination and degradation of occludin during permanent brain ischemia, suggesting that γ-secretase may represent a novel therapeutic target for preventing neurovascular damage.

Topics: Amyloid Precursor Protein Secretases; Analysis of Variance; Animals; Blood-Brain Barrier; Dipeptides; Disease Models, Animal; Endothelium, Vascular; Enzyme Inhibitors; Gene Expression Regulation; Immunoprecipitation; Infarction, Middle Cerebral Artery; Male; Occludin; Permeability; Platelet Endothelial Cell Adhesion Molecule-1; Rats; Rats, Sprague-Dawley; Tight Junctions; Time Factors; Ubiquitin-Protein Ligases; Ubiquitination; Zonula Occludens-1 Protein

Notch1 signaling controls the cardiac adaptation to stress. We therefore aimed to validate whether olmesartan, a widely used angiotensin II type 1 receptor blocker, ameliorates cardiac remodeling and dysfunction via delta-like ligand 4 (DLL4)/Notch1 pathway in mice with chronic pressure overload.. Cardiac pressure overload was produced by transverse aortic constriction (TAC). A total of 35 wide-type C57BL/6J mice were randomly divided into sham group, TAC group, TAC + olmesartan group, and TAC + olmsartan + DAPT group (DAPT: γ-secretase inhibitor, Notch signaling inhibitor). Saline (10 mL·kg(-1)·d(-1)) or the same volume of olmesartan liquor (3 mg·kg(-1) d(-1)) was administered by gavage, and DAPT (10 μmole·kg(-1)·d(-1)) by peritoneal injection. After 28 days of treatment, cardiac hemodynamics, echocardiography, and histology were evaluated, followed by quantitative polymerase chain reaction of fetal gene (ANP and SAA) expression. Notch1-related proteins and ERK1/2 were examined by western blot, and the serum level of angiotensin II was determined by means of enzyme-linked immunosorbent assay kits.. Persistent pressure overload-induced left ventricular hypertrophy, dysfunction, fibrosis, and microcirculation dysfunction, together with the upregulation of angiotensin II, ERK1/2, and fetal gene expression. By the activation of DLL4/Notch1, olmesartan decreased left ventricular hypertrophy and fibrosis, preserved cardiac function, and improved capillary density and coronary perfusion. All these curative effects were suppressed by pharmacological blockade of Notch signaling with DAPT.. Our findings identify a heretofore unknown pharmacological mechanism that olmesartan improves cardiac remodeling and function via DLL4/Notch1 pathway activation in mice with chronic pressure overload, which may present a new therapeutic target for hypertension.

Topics: Adaptor Proteins, Signal Transducing; Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Calcium-Binding Proteins; Dipeptides; Disease Models, Animal; Fibrosis; Hypertrophy, Left Ventricular; Imidazoles; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Microcirculation; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Receptor, Notch1; Tetrazoles; Up-Regulation; Ventricular Dysfunction, Left; Ventricular Remodeling

The Notch pathway is an evolutionarily conserved signaling cascade that is critical in kidney development and has also been shown to play a pathogenetic role in a variety of kidney diseases. We have previously shown that the Notch signaling pathway is activated in human immunodeficiency virus-associated nephropathy (HIVAN) as well as in a rat model of the disease. In this study, we examined Notch signaling in the well established Tg26 mouse model of HIVAN. Notch signaling components were distinctly upregulated in the kidneys of these mice as well as in immortalized podocytes derived from these mice. Notch1 and Notch4 were upregulated in the Tg26 glomeruli, and Notch4 was also expressed in tubules. Notch ligands Jagged1, Jagged2, Delta-like1, and Delta-like 4 were all upregulated in the tubules of Tg26 mice, but glomeruli showed minimal expression of Notch ligands. To examine a potential pathogenetic role for Notch in HIVAN, Tg26 mice were treated with GSIXX, a gamma secretase inhibitor that blocks Notch signaling. Strikingly, GSIXX treatment resulted in significant improvement in both histological kidney injury scores and renal function. GSIXX-treated Tg26 mice also showed diminished podocyte proliferation and dedifferentiation, cellular hallmarks of the disease. Moreover, GSIXX blocked podocyte proliferation in vitro induced by HIV proteins Nef and Tat. These studies suggest that Notch signaling can promote HIVAN progression and that Notch inhibition may be a viable treatment strategy for HIVAN.

Topics: AIDS-Associated Nephropathy; Amyloid Precursor Protein Secretases; Animals; Cell Dedifferentiation; Cell Division; Cell Line, Transformed; Dibenzazepines; Dipeptides; Disease Models, Animal; Disease Progression; Humans; Kidney; Ligands; Mice; Mice, Inbred Strains; Mice, Transgenic; Podocytes; Proto-Oncogene Proteins; Receptor, Notch1; Receptor, Notch4; Receptors, Notch; Signal Transduction; Up-Regulation

Treatment with DAPT, an inhibitor of the Notch-activating enzyme, γ-secretase is known to reduce damage to ischemic brain. However, the molecular mechanisms supporting this therapeutic effect are not fully understood. Here we demonstrated that Notch/RBP-J signaling is activated in NG2(+) glial progenitors and reactive astrocytes such as GFAP(+) cells, Nestin(+) cells and RC2(+) cells, using Notch/RBP-J signaling reporter mice. 3-day DAPT treatment reduced the number of reactive astrocytes but not NG2(+) glial progenitors. BrdU labeling experiments have shown that this reduction was due to decreased proliferation of reactive astrocytes. DAPT inhibited nuclear-translocation of Olig2, which is indispensable for proliferation and differentiation of reactive astrocytes. These findings suggest that Notch signaling might promote proliferation and differentiation of reactive astrocytes through the regulation of nucleo-cytoplasmic translocation of Olig2.

Topics: Amyloid Precursor Protein Secretases; Animals; Astrocytes; Basic Helix-Loop-Helix Transcription Factors; Cell Differentiation; Cell Nucleus; Cell Proliferation; Cytoplasm; Dipeptides; Disease Models, Animal; Mice; Mice, Transgenic; Nerve Tissue Proteins; Neurogenesis; Neuroglia; Oligodendrocyte Transcription Factor 2; Protein Transport; Receptors, Notch; Signal Transduction; Stroke

Activin receptor-like kinase 1 (ALK1) is an endothelial-specific member of the TGF-β/BMP receptor family that is inactivated in patients with hereditary hemorrhagic telangiectasia (HHT). How ALK1 signaling regulates angiogenesis remains incompletely understood. Here we show that ALK1 inhibits angiogenesis by cooperating with the Notch pathway. Blocking Alk1 signaling during postnatal development in mice leads to retinal hypervascularization and the appearance of arteriovenous malformations (AVMs). Combined blockade of Alk1 and Notch signaling further exacerbates hypervascularization, whereas activation of Alk1 by its high-affinity ligand BMP9 rescues hypersprouting induced by Notch inhibition. Mechanistically, ALK1-dependent SMAD signaling synergizes with activated Notch in stalk cells to induce expression of the Notch targets HEY1 and HEY2, thereby repressing VEGF signaling, tip cell formation, and endothelial sprouting. Taken together, these results uncover a direct link between ALK1 and Notch signaling during vascular morphogenesis that may be relevant to the pathogenesis of HHT vascular lesions.

Topics: Activin Receptors, Type I; Activin Receptors, Type II; Animals; Arteriovenous Malformations; Basic Helix-Loop-Helix Transcription Factors; Cell Cycle Proteins; Dipeptides; Disease Models, Animal; Growth Differentiation Factor 2; Growth Differentiation Factors; Humans; Mice; Mice, Inbred C57BL; Neovascularization, Physiologic; Receptors, Notch; Repressor Proteins; Retina; Signal Transduction; Smad Proteins; Telangiectasia, Hereditary Hemorrhagic; Vascular Endothelial Growth Factors

The Notch signaling pathway has been shown to be involved in the development of the nervous system. Recent studies showed that Notch receptors and ligands are also expressed in the nervous system of adult animals. However, whether the Notch signaling pathway has a function in adults is not fully understood. The present study is designed to investigate the function of the Notch signaling pathway in nociceptive transmission, especially during neuropathic pain in adult rats.. We found that the Notch intracellular domain (NICD) is expressed in the DRG (Dorsal Root Ganglia), sciatic nerve and spinal cord in normal rats, and is upregulated in the sciatic nerve and spinal cord after spared nerve injury (SNI). Moreover, we used the γ-secretase (a key enzyme of the Notch signaling pathway) inhibitor DAPT to observe the effect of the Notch signaling pathway after SNI. We found that intrathecal DAPT significantly increased paw withdrawal thermal latency and mechanical threshold. Mechanical hyperalgesia occurring after SNI could be significantly reversed by DAPT in a dose-dependent manner.. These results suggest that the Notch signaling pathway participates in the induction and maintenance of neuropathic pain, which indicates that the Notch pathway maybe a potential drug target for neuropathic pain treatment.

Topics: Animals; Constriction; Dipeptides; Disease Models, Animal; Ganglia, Spinal; Hyperalgesia; Male; Neuralgia; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Receptors, Notch; Sciatic Nerve; Signal Transduction; Spinal Cord; Temperature

Neuroligin (NLG), a postsynaptic adhesion molecule, is involved in the formation of synapses by binding to a cognate presynaptic ligand, neurexin. Here we report that neuroligin-1 (NLG1) undergoes ectodomain shedding at the juxtamembrane stalk region to generate a secreted form of NLG1 and a membrane-tethered C-terminal fragment (CTF) in adult rat brains in vivo as well as in neuronal cultures. Pharmacological and genetic studies identified ADAM10 as the major protease responsible for NLG1 shedding, the latter being augmented by synaptic NMDA receptor activation or interaction with soluble neurexin ligands. NLG1-CTF was subsequently cleaved by presenilin/γ-secretase. Secretion of soluble NLG1 was significantly upregulated under a prolonged epileptic seizure condition, and inhibition of NLG1 shedding led to an increase in numbers of dendritic spines in neuronal cultures. Collectively, neuronal activity-dependent proteolytic processing of NLG1 may negatively regulate the remodeling of spines at excitatory synapses.

Topics: ADAM Proteins; ADAM10 Protein; Amyloid Precursor Protein Secretases; Animals; Animals, Newborn; Biotinylation; Calcium-Binding Proteins; Cell Adhesion Molecules, Neuronal; Cells, Cultured; Cerebellum; Dendritic Spines; Dipeptides; Disease Models, Animal; Embryo, Mammalian; Enzyme Inhibitors; Gene Expression Regulation; Green Fluorescent Proteins; Hippocampus; Humans; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Knockout; Muscarinic Agonists; Nerve Tissue Proteins; Neural Cell Adhesion Molecules; Neurons; Organ Culture Techniques; Pilocarpine; Proteolysis; Rats; Rats, Wistar; RNA Interference; Status Epilepticus; Synaptosomes; Transfection

Activation of inflammatory pathways plays a critical role in the development of abdominal aortic aneurysms (AAA). Notch1 signaling is a significant regulator of the inflammatory response; however, its role in AAA is unknown.. In an angiotensin II-induced mouse model of AAA, activation of Notch1 signaling was observed in the aortic aneurysmal tissue of Apoe(-/-) mice, and a similar activation of Notch1 was observed in aneurysms of humans undergoing AAA repair. Notch1 haploinsufficiency significantly reduced the incidence of AAA in Apoe(-/-) mice in response to angiotensin II. Reconstitution of bone marrow-derived cells from Notch1(+/-);Apoe(-/-) mice (donor) in lethally irradiated Apoe(-/-) mice (recipient) decreased the occurrence of aneurysm. Flow cytometry and immunohistochemistry demonstrated that Notch1 haploinsufficiency prevented the influx of inflammatory macrophages at the aneurysmal site by causing defects in macrophage migration and proliferation. In addition, there was an overall reduction in the inflammatory burden in the aorta of the Notch1(+/-);Apoe(-/-) mice compared with the Apoe(-/-) mice. Last, pharmacological inhibition of Notch1 signaling also prevented AAA formation and progression in Apoe(-/-) mice.. Our data suggest that decreased levels of Notch1 protect against the formation of AAA by preventing macrophage recruitment and attenuating the inflammatory response in the aorta.

Topics: Angiotensin II; Animals; Aortic Aneurysm, Abdominal; Apolipoproteins E; Arteritis; Dipeptides; Disease Models, Animal; Haploinsufficiency; Humans; Macrophages; Male; Mice; Mice, Knockout; Receptor, Notch1; Signal Transduction

Pulmonary hypertension (PH) is a fatal disease that lacks an effective therapy. Notch signaling pathway plays a crucial role in the angiogenesis and vascular remodeling. However, its roles in vascular remodeling in PH have not been well studied. In the current study, using hypoxia-induced PH model in rat, we examined the expression of Notch and its downstream factors. Then, we used vessel strip culture system and γ-secretase inhibitor DAPT, a Notch signaling inhibitor to determine the effect of Notch signaling in vascular remodeling and its potential therapeutic value. Our results indicated that Notch 1-4 were detected in the lung tissue with variable levels in different cell types such as smooth muscle cells and endothelial cells of pulmonary artery, bronchia, and alveoli. In addition, following the PH induction, all of Notch1, Notch3, Notch4 receptor, and downstream factor, HERP1 in pulmonary arteries, mRNA expressions were increased with a peak at 1-2 weeks. Furthermore, the vessel wall thickness from rats with hypoxia treatment increased after cultured for 8 days, which could be decreased approximately 30% by DAPT, accompanied with significant increase of expression level of apoptotic factors (caspase-3 and Bax) and transformation of vascular smooth muscle cell (VSMC) phenotype from synthetic towards contractile. In conclusion, the current study suggested Notch pathway plays an important role in pulmonary vascular remodeling in PH and targeting Notch signaling pathway could be a valuable approach to design new therapy for PH.

Topics: Animals; Apoptosis; Blood Vessels; Cell Proliferation; Cells, Cultured; Dipeptides; Disease Models, Animal; Gene Expression Regulation; Hypertension, Pulmonary; Hypoxia; Immunohistochemistry; In Vitro Techniques; Lung; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phenotype; Rats; Rats, Wistar; Receptors, Notch; Reproducibility of Results; Signal Transduction; Staining and Labeling

Synaptic loss is the best pathological correlate of the cognitive decline in Alzheimer's disease; however, the molecular mechanisms underlying synaptic failure are unknown. We found a non-apoptotic baseline caspase-3 activity in hippocampal dendritic spines and an enhancement of this activity at the onset of memory decline in the Tg2576-APPswe mouse model of Alzheimer's disease. In spines, caspase-3 activated calcineurin, which in turn triggered dephosphorylation and removal of the GluR1 subunit of AMPA-type receptor from postsynaptic sites. These molecular modifications led to alterations of glutamatergic synaptic transmission and plasticity and correlated with spine degeneration and a deficit in hippocampal-dependent memory. Notably, pharmacological inhibition of caspase-3 activity in Tg2576 mice rescued the observed Alzheimer-like phenotypes. Our results identify a previously unknown caspase-3-dependent mechanism that drives synaptic failure and contributes to cognitive dysfunction in Alzheimer's disease. These findings indicate that caspase-3 is a potential target for pharmacological therapy during early disease stages.

Topics: Alzheimer Disease; Animals; Calcineurin; Caspase 3; Caspase Inhibitors; Dendritic Spines; Dipeptides; Disease Models, Animal; Gene Expression Regulation; Hippocampus; Long-Term Synaptic Depression; Memory Disorders; Mice; Mice, Transgenic; Nerve Degeneration; Oligopeptides; Polyglutamic Acid; Receptors, AMPA; Synaptic Transmission

Wet age-related macular degeneration (AMD), which accounts for most AMD-related vision loss, is characterized by choroidal neovascularization (CNV). The underlying mechanism of CNV is poorly understood, but evidence indicates pathologic recruitment of normal angiogenic signaling pathways such as the VEGF pathway. Recent evidence suggests that the VEGF pathway regulates angiogenesis in concert with Notch signaling. Here, the authors examined the role of Notch signaling in CNV in the backdrop of Notch signaling-mediated regulation of retinal angiogenesis.. Choroid sclera complexes, after laser-induced CNV, were examined for changes in CNV lesion volume and in proangiogenic and antiangiogenic gene expression after perturbation in Notch signaling. Retinal vessels and angiogenic gene expression in retinal endothelial cells were analyzed in postnatal rats after perturbations in Notch signaling. Notch signaling was activated and inhibited by intravitreal or systemic injection of Jagged1 peptide and gamma secretase inhibitor DAPT, respectively.. The authors demonstrated that activation of the canonical Notch pathway reduced the volume of CNV lesions as it attenuated the development of postnatal retinal vasculature. In contrast, inhibition of the Notch pathway exacerbated CNV lesions as it led to the development of hyperdense retinal vasculature. The authors also identified genes associated with proangiogenesis (Vegfr2, Ccr3, and Pdgfb) and antiangiogenesis (Vegfr1 and Unc5b) as targets of Notch signaling-mediated vascular homeostasis, the disruption of which might underlie CNV.. This study suggests that Notch signaling is a key regulator of CNV and thus a molecular target for therapeutic intervention in wet AMD.

Topics: Animals; Calcium-Binding Proteins; Choroidal Neovascularization; Dipeptides; Disease Models, Animal; Fluorescein Angiography; Gene Expression Regulation; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; Membrane Proteins; Proto-Oncogene Proteins c-sis; Rats; Rats, Sprague-Dawley; Receptors, CCR3; Receptors, Notch; Retinal Neovascularization; Retinal Vessels; Reverse Transcriptase Polymerase Chain Reaction; Serrate-Jagged Proteins; Signal Transduction; Vascular Endothelial Growth Factor Receptor-2; Wet Macular Degeneration

Inflammatory bowel disease increases the risks of colon cancer and colitis-associated cancer (CAC). Epithelial cell-derived matrix metalloproteinase (MMP)-9 mediates inflammation during acute colitis and the cleavage and activation of the transcription factor Notch1, which prevents differentiation of progenitor cells into goblet cells. However, MMP-9 also protects against the development of CAC and acts as a tumor suppressor. We investigated the mechanisms by which MMP-9 protects against CAC in mice.. C57/B6 wild-type mice were given a single dose of azoxymethane and 2 cycles of dextran sulfate sodium (DSS). Mice were also given the γ-secretase inhibitor difluorophenacetyl-l-alanyl-S-phenylglycine t-butyl ester (DAPT) or dimethyl sulfoxide (control) during each DSS cycle; they were killed on day 56. We analyzed embryonic fibroblasts isolated from wild-type and MMP-9-/- mice and HCT116 cells that were stably transfected with MMP-9.. Wild-type mice were more susceptible to CAC following inhibition of Notch1 by DAPT, shown by increased numbers of tumors and level of dysplasia compared with controls. Inhibition of Notch1 signaling significantly reduced protein levels of active Notch1, p53, p21WAF1/Cip1, Bax-1, active caspase-3, as well as apoptosis, compared with controls. Similar results were observed in transgenic HCT116 cells and embryonic fibroblasts from MMP-9-/- mice on γ-radiation-induced damage of DNA.. MMP-9 mediates Notch1 signaling via p53 to regulate apoptosis, cell cycle arrest, and inflammation. By these mechanisms, it might prevent CAC.

Topics: Amyloid Precursor Protein Secretases; Animals; Apoptosis; Azoxymethane; Caspase 3; Colitis; Colon; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cytokines; Dextran Sulfate; Dipeptides; Disease Models, Animal; DNA Damage; Enzyme Inhibitors; Fibroblasts; Gamma Rays; HCT116 Cells; Humans; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Mice, Knockout; Receptor, Notch1; RNA, Messenger; Signal Transduction; Time Factors; Transfection; Tumor Suppressor Protein p53

Glomerular diseases account for 90% of end-stage kidney disease. Podocyte loss is a common determining factor for the progression toward glomerulosclerosis. Mature podocytes cannot proliferate, but recent evidence suggests that they can be replaced by renal progenitors localized within the Bowman's capsule. Here, we demonstrate that Notch activation in human renal progenitors stimulates entry into the S-phase of the cell cycle and cell division, whereas its downregulation is required for differentiation toward the podocyte lineage. Indeed, a persistent activation of the Notch pathway induced podocytes to cross the G(2)/M checkpoint, resulting in cytoskeleton disruption and death by mitotic catastrophe. Notch expression was virtually absent in the glomeruli of healthy adult kidneys, while a strong upregulation was observed in renal progenitors and podocytes in patients affected by glomerular disorders. Accordingly, inhibition of the Notch pathway in mouse models of focal segmental glomerulosclerosis ameliorated proteinuria and reduced podocyte loss during the initial phases of glomerular injury, while inducing reduction of progenitor proliferation during the regenerative phases of glomerular injury with worsening of proteinuria and glomerulosclerosis. Taken altogether, these results suggest that the severity of glomerular disorders depends on the Notch-regulated balance between podocyte death and regeneration provided by renal progenitors.

Topics: Animals; Case-Control Studies; Cell Cycle; Cell Death; Cell Differentiation; Cell Lineage; Cell Proliferation; Cells, Cultured; Dipeptides; Disease Models, Animal; Dose-Response Relationship, Drug; Doxorubicin; Female; Glomerulosclerosis, Focal Segmental; Humans; Lupus Nephritis; Mice; Mice, SCID; Podocytes; Proteinuria; Receptors, Notch; Severity of Illness Index; Stem Cells; Time Factors; Transfection

Human papillomaviruses (HPV) are common sexually transmitted pathogens that predispose women to cervical and other anogenital cancers. HPV vaccines can prevent infection by some but not other sexually transmitted HPVs but are too costly for use in much of the world at greatest risk to HPV-associated cancers. Microbicides provide an inexpensive alternative to vaccines. In a high throughput screen, drugs that inhibit the cellular protein complex known as gamma secretase were identified as potential HPV microbicides. gamma Secretase inhibitors (GSIs) inhibited the infectivity of HPV pseudoviruses both in human keratinocytes and in mouse cells, with IC(50) values in the picomolar to the nanomolar range. Using a mouse model, we observed that a GSI could inhibit HPV infection to the same degree as its effectiveness in inhibiting gamma secretase activity in vivo. We conclude that gamma secretase activity is required for HPV infection and that GSIs are effective microbicides against anogenital HPVs.

Topics: Amyloid Precursor Protein Secretases; Animals; Anti-Infective Agents; Carbamates; Cell Line; Cells, Cultured; Dipeptides; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Genital Diseases, Female; Human papillomavirus 16; Humans; Keratinocytes; Mice; Papillomavirus Infections; Treatment Outcome

Notch receptor signaling is implicated in controlling smooth muscle cell proliferation and in maintaining smooth muscle cells in an undifferentiated state. Pulmonary arterial hypertension is characterized by excessive vascular resistance, smooth muscle cell proliferation in small pulmonary arteries, leading to elevation of pulmonary vascular resistance, right ventricular failure and death. Here we show that human pulmonary hypertension is characterized by overexpression of NOTCH3 in small pulmonary artery smooth muscle cells and that the severity of disease in humans and rodents correlates with the amount of NOTCH3 protein in the lung. We further show that mice with homozygous deletion of Notch3 do not develop pulmonary hypertension in response to hypoxic stimulation and that pulmonary hypertension can be successfully treated in mice by administration of N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a gamma-secretase inhibitor that blocks activation of Notch3 in smooth muscle cells. We show a mechanistic link from NOTCH3 receptor signaling through the Hairy and enhancer of Split-5 (HES-5) protein to smooth muscle cell proliferation and a shift to an undifferentiated smooth muscle cell phenotype. These results suggest that the NOTCH3-HES-5 signaling pathway is crucial for the development of pulmonary arterial hypertension and provide a target pathway for therapeutic intervention.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Proliferation; Dipeptides; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation; Humans; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Lung; Mice; Mice, Knockout; Microscopy, Electron, Transmission; Myocytes, Smooth Muscle; Pulmonary Artery; Rats; Receptor, Notch3; Receptors, Notch; Repressor Proteins; RNA, Messenger; Signal Transduction; Time Factors

Accumulation of amyloid beta (Abeta) in the brain is believed to represent one of the earliest events in the Alzheimer disease process. Abeta is generated from amyloid precursor protein after sequential cleavage by beta- and gamma-secretase. Alternatively, alpha-secretase cleaves within the Abeta sequence, thus, precluding the formation of Abeta. A lot of research has focused on Abeta production, while less is known about the non-amyloidogenic pathway. We have previously shown that Abeta is present in human cerebrospinal fluid (CSF) as several shorter C-terminal truncated isoforms (e.g. Abeta1-15 and Abeta1-16), and that the levels of these shorter isoforms are elevated in media from cells that have been treated with gamma-secretase inhibitors.. To explore the effect of N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT), a gamma-secretase-inhibitor, treatment on the Abeta isoform pattern in brain tissue and CSF from Tg2576 mice.. Immunoprecipitation using the anti-Abeta antibodies 6E10 and 4G8 was combined with either matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or nanoflow liquid chromatography and tandem mass spectrometry.. All fragments longer than and including Abeta1-17 displayed a tendency towards decreased levels upon gamma-secretase inhibition, whereas Abeta1-15 and Abeta1-16 indicated slightly elevated levels during treatment.. These data suggest that Abeta1-15 and Abeta1-16 may be generated through a third metabolic pathway independent of gamma-secretase, and that these Abeta isoforms may serve as biomarkers for secretase inhibitor treatment.

Topics: Age Factors; Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Brain; Dipeptides; Disease Models, Animal; Enzyme Inhibitors; Humans; Immunoprecipitation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Peptide Fragments; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

SAR on HTS hits 1 and 2 led to the potent, Notch-1-sparing GSI 9, which lowered brain Abeta in Tg2576 mice at 100 mg/kg po. Converting the metabolically labile methyl groups in 9 to trifluoromethyl groups afforded the more stable analogue 10, which had improved in vivo potency. Further side chain modification afforded the potent Notch-1-sparing GSI begacestat (5), which was selected for development for the treatment of Alzheimer's disease.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Animals; Crystallography, X-Ray; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Discovery; Enzyme Inhibitors; Mice; Mice, Transgenic; Models, Molecular; Molecular Conformation; Receptor, Notch1; Stereoisomerism; Structure-Activity Relationship; Sulfonamides; Thiophenes

Patients with autoimmune lymphoproliferative syndrome (ALPS) and systemic lupus erythematosis (SLE) have T-cell dysregulation and produce abnormal, activated T lymphocytes and an atypical peripheral T-cell population, termed double negative T cells (DNTs). T-cell functions, including DNT transition in T-cell development and T-cell activation, are critically dependent on Notch signaling. We hypothesized that inhibiting Notch signaling would be effective in ALPS and SLE by reducing the production of abnormal DNTs and by blocking aberrant T-cell activation. We tested this hypothesis using murine models of ALPS and SLE. Mice were randomized to treatment with the notch pathway inhibitor (gamma-secretase inhibitor), N-S-phenyl-glycine-t-butyl ester (DAPT), or vehicle control. Response to treatment was assessed by measurement of DNTs in blood and lymphoid tissue, by monitoring lymph node and spleen size with ultrasound, by quantifying cytokines by bead-array, by ELISA for total IgG and anti-double-stranded DNA (dsDNA) specific antibodies, and by histopathologic assessment for nephritis. We found a profound and statistically significant decrease in all disease parameters, comparing DAPT-treated mice to controls. Using a novel dosing schema, we avoided the reported toxicities of gamma-secretase inhibitors. Inhibiting the Notch signaling pathway may thus present an effective, novel, and well-tolerated treatment for autoimmune and lymphoproliferative diseases.

Topics: Animals; Antibodies, Antinuclear; Dipeptides; Disease Models, Animal; Drug Evaluation, Preclinical; Enzyme Inhibitors; Humans; Immunoglobulin G; Lupus Erythematosus, Systemic; Lymph Nodes; Lymphocyte Activation; Lymphoproliferative Disorders; Mice; Mice, Inbred CBA; Mice, Inbred MRL lpr; Nephritis; Random Allocation; Receptors, Notch; Signal Transduction; Spleen; T-Lymphocytes; Ultrasonography

Considerable effort has been made to develop drugs that delay or prevent neurodegeneration. These include inhibitors of Abeta-generating proteases for the treatment of Alzheimer's disease. Testing the amyloid hypothesis in vivo requires molecules that are capable of entering the CNS and that produce a substantial reduction in brain Abeta levels. Plaque-developing APP transgenic mice are currently widely used as an in vivo model of choice as these animals produce readily measurable amounts of human Abeta. They are very useful in the testing of a variety of amyloid-lowering approaches but their use for compound screening is often limited by their cost. Transgenic animals also require extensive, time-consuming breeding programs and can show high inter-animal differences in the expression level of the transgene. Hence, we considered it important to develop and characterize a new and simple non-transgenic animal model for testing Abeta modulation. For this purpose, Wild-type adult Sprague Dawley rats were treated with DAPT, a functional gamma-secretase inhibitor, and the Abeta40 and Abeta42 levels in brain-tissue and body fluids were assessed. We showed that DAPT, given orally, significantly lowered Abeta40 and Abeta42 peptide levels in brain extract, CSF, and the plasma dose- and time-dependently. We can conclude that our data establish the usefulness of the wild-type rat model for testing small-molecule inhibitors of Abeta production.

Topics: Administration, Oral; Alzheimer Disease; Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Brain; Dipeptides; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Endopeptidases; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Female; Humans; Male; Peptide Fragments; Rats; Rats, Sprague-Dawley; Time Factors

Chronic neuroinflammation plays a prominent role in the progression of Alzheimer's disease. Reactive microglia and astrocytes are observed within the hippocampus during the early stages of the disease. Epidemiological findings suggest that anti-inflammatory therapies may slow the onset of Alzheimer's disease. Chemokine receptor 5 (CCR5) up-regulation may influence the recruitment and accumulation of glia near senile plaques; activated microglia express CCR5 and reactive astrocytes express chemokines. We have previously shown that neuroinflammation induced by chronic infusion of lipopolysaccharide into the 4th ventricle reproduces many of the behavioral, neurochemical, electrophysiological and neuropathological changes associated with Alzheimer's disease. The current study investigated the ability of D-Ala-peptide T-amide (DAPTA), a chemokine receptor 5 chemokine receptor antagonist of monocyte chemotaxis, to influence the consequences of chronic infusion of lipopolysaccharide. DAPTA (0.01 mg/kg, s.c., for 14 days) dramatically reduced the number of activated microglia and astrocytes, as compared with lipopolysaccharide-infused rats treated with vehicle. DAPTA treatment also reduced the number of immunoreactive cells expressing nuclear factor kappa binding protein, a prominent component of the proinflammatory cytokine signaling pathway. The present study suggests that DAPTA and other CCR5 antagonists may attenuate critical aspects of the neuroinflammation associated with Alzheimer's disease.

Topics: Animals; Astrocytes; CCR5 Receptor Antagonists; Dipeptides; Disease Models, Animal; Hippocampus; Inflammation; Lipopolysaccharides; Microglia; Microscopy, Confocal; Rats

Converging lines of evidence implicate the beta-amyloid peptide (Ass) as causative in Alzheimer's disease. We describe a novel class of compounds that reduce A beta production by functionally inhibiting gamma-secretase, the activity responsible for the carboxy-terminal cleavage required for A beta production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon A beta production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP). Oral administration of one of these compounds, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, to mice transgenic for human APP(V717F) reduces brain levels of Ass in a dose-dependent manner within 3 h. These studies represent the first demonstration of a reduction of brain A beta in vivo. Development of such novel functional gamma-secretase inhibitors will enable a clinical examination of the A beta hypothesis that Ass peptide drives the neuropathology observed in Alzheimer's disease.

Topics: Administration, Oral; Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Brain; Cells, Cultured; Dipeptides; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Endopeptidases; Enzyme Inhibitors; Female; Humans; Injections, Subcutaneous; Kidney; Male; Mice; Mice, Transgenic; Neurons; Peptide Fragments