Gamma-secretase Inhibitor DAPT Research Articles

The reciprocal regulation between mitochondrial-associated membranes and Notch signaling in skeletal muscle atrophy

Skeletal muscle atrophy and the inhibition of muscle regeneration are known to occur as a natural consequence of aging, yet the underlying mechanisms that lead to these processes in atrophic myofibers remain largely unclear. Our research has revealed that the maintenance of proper mitochondrial-associated endoplasmic reticulum membranes (MAM) is vital for preventing skeletal muscle atrophy in microgravity environments. We discovered that the deletion of the mitochondrial fusion protein Mitofusin2 (MFN2), which serves as a tether for MAM, in human induced pluripotent stem (iPS) cells or the reduction of MAM in differentiated myotubes caused by microgravity interfered with myogenic differentiation process and an increased susceptibility to muscle atrophy, as well as the activation of the Notch signaling pathway. The atrophic phenotype of differentiated myotubes in microgravity and the regenerative capacity of Mfn2-deficient muscle stem cells in dystrophic mice were both ameliorated by treatment with the gamma-secretase inhibitor DAPT. Our findings demonstrate how the orchestration of mitochondrial morphology in differentiated myotubes and regenerating muscle stem cells plays a crucial role in regulating Notch signaling through the interaction of MAM.

The reciprocal regulation between mitochondrial-associated membranes and Notch signaling in skeletal muscle atrophy

Skeletal muscle atrophy and the inhibition of muscle regeneration are known to occur as a natural consequence of aging, yet the underlying mechanisms that lead to these processes in atrophic myofibers remain largely unclear. Our research has revealed that the maintenance of proper mitochondrial-associated endoplasmic reticulum membranes (MAM) is vital for preventing skeletal muscle atrophy in microgravity environments. We discovered that the deletion of the mitochondrial fusion protein Mitofusin2 (MFN2), which serves as a tether for MAM, in human induced pluripotent stem (iPS) cells or the reduction of MAM in differentiated myotubes caused by microgravity interfered with myogenic differentiation process and an increased susceptibility to muscle atrophy, as well as the activation of the Notch signaling pathway. The atrophic phenotype of differentiated myotubes in microgravity and the regenerative capacity of Mfn2-deficient muscle stem cells in dystrophic mice were both ameliorated by treatment with the gamma-secretase inhibitor DAPT. Our findings demonstrate how the orchestration of mitochondrial morphology in differentiated myotubes and regenerating muscle stem cells plays a crucial role in regulating Notch signaling through the interaction of MAM.

Sustained notch signaling inhibition with a gamma-secretase inhibitor prevents traumatic heterotopic ossification

BackgroundTraumatic heterotopic ossification (THO) is a devastating sequela following traumatic injuries and orthopedic surgeries. To date, the exact molecular mechanism of THO formation is still unclear, which hinders the development of effective treatments. The process of THO formation is believed to recapitulate a series of spatiotemporal cellular and signaling events that occur during skeletal development. The Notch signaling pathway is a critical genetic regulator in embryological bone development and fracture healing. However, few data are available concerning whether Notch signaling regulates THO development and maturation. MethodsWe firstly detected the expressions of Notch target genes in both mouse and human THO samples with quantitative RT-PCR and immunohistochemistry (IHC). Then, tissue-resident mesenchymal progenitor cells (TMPCs) were isolated, and the abilities of the proliferation and osteogenic and chondrogenic differentiation of TMPCs were examined under the intervention of the gamma-secretase inhibitor-DAPT at different time points. Finally, DAPT was also administrated in THO mice by burn and Achilles tenotomy injury, and ectopic cartilage and bone formation were monitored by histology and micro-CT. ResultsSeveral Notch target genes were upregulated in both mouse and human THO tissues. Sustained Notch signaling inhibition by DAPT reduced proliferation, osteogenic and chondrogenic differentiation of TMPCs in a time-dependent manner. Moreover, DAPT administration within 3 weeks could inhibit ectopic cartilage and bone formation in a mouse THO model without affecting the total body bone mass. ConclusionsThe Notch signaling serves as an important therapeutic target during THO formation. And sustained gamma-secretase inhibition by DAPT has great potential in repressing chondrogenic and osteogenic differentiation of TMPCs, as well as inhibited ectopic cartilage and bone formation in vivo. The translational potential of this articleSustained Notch inhibition via systemic DAPT (or other similar gamma-secretase inhibitors) administration has promising clinical utility for inhibiting THO formation, providing new insight into THO prophylaxis and treatment.

In silico identification of potential γ-secretase inhibitor of marine-algal origin: an anticancer intervention

ABSTRACT Gamma secretase (GS) activates notch signalling pathway (NSP) by liberating the truncated notch intracellular domain (NICD). The NSP is associated with the cancer development and progression, which makes GS a potential therapeutic target. Now day’s marine compounds emerged as a major source of bioactive entity. The NSP inhibition potential of marine-algal compounds has not yet been studied. Thus, in the present study, we have used molecular docking, molecular dynamics (MD) simulations, principal component analysis (PCA) and free energy and binding energy calculations to identify the potential GS inhibitors of marine-algal origin. Laminarin showed better docking score (−12.72) compared to the known GS inhibitor DAPT (−9.2). Laminarin formed H-Bond interaction with the Asp257 and Asp385 required for the catalytic cleavage activity of gamma-secretase. It potentially stabilised the structural parameters (RMSD, RMSF, Rg and SASA) of GS catalytic subunit compared to DAPT during the MD simulation. The PCA and free energy calculation revealed conformationally and energetically stable Laminarin–GS complex formation. Laminarin showed lower binding energy (−44.75 kcal/mol) with GS catalytic subunit than DAPT (−20.92 kcal/mol). In conclusion, the present study provides a marine-algal compound as a novel potential GS inhibitor, which requires further validation in experimental model.

Notch-triggered maladaptation of liver sinusoidal endothelium aggravates nonalcoholic steatohepatitis through endothelial nitric oxide synthase.

Although NASH can lead to severe clinical consequences, including cirrhosis and hepatocellular carcinoma, no effective treatment is currently available for this disease. Increasing evidence indicates that LSECs play a critical role in NASH pathogenesis; however, the mechanisms involved in LSEC-mediated NASH remain to be fully elucidated. In the current study, we found that LSEC homeostasis was disrupted and LSEC-specific gene profiles were altered in methionine-choline-deficient (MCD) diet-induced NASH mouse models. Importantly, Notch signaling was found to be activated in LSECs of NASH mice. To then investigate the role of endothelial Notch in NASH progression, we generated mouse lines with endothelial-specific Notch intracellular domain (NICD) overexpression or RBP-J knockout to respectively activate or inhibit Notch signaling in endothelial cells. Notably, endothelial-specific overexpression of the NICD accelerated LSEC maladaptation and aggravated NASH, whereas endothelial cell-specific inhibition of Notch signaling restored LSEC homeostasis and improved NASH phenotypes. Furthermore, we demonstrated that endothelial-specific Notch activation exacerbated NASH by inhibiting endothelial nitric oxide synthase (eNOS) transcription, whereas administration of the pharmacological eNOS activator YC-1 alleviated hepatic steatosis and lipid accumulation resulting from Notch activation. Finally, to explore the therapeutic potential of using Notch inhibitors in NASH treatment, we applied two gamma-secretase inhibitors-DAPT and LY3039478-in an MCD diet-induced mouse model of NASH, and found that both inhibitors effectively ameliorated hepatic steatosis, inflammation, and liver fibrosis. Endothelial-specific Notch activation triggered LSEC maladaptation and exacerbated NASH phenotypes in an eNOS-dependent manner. Genetic and pharmacological inhibition of Notch signaling effectively restored LSEC homeostasis and ameliorated NASH progression.

Modelling of primary ciliary dyskinesia using patient-derived airway organoids.

Patient‐derived human organoids can be used to model a variety of diseases. Recently, we described conditions for long‐term expansion of human airway organoids (AOs) directly from healthy individuals and patients. Here, we first optimize differentiation of AOs towards ciliated cells. After differentiation of the AOs towards ciliated cells, these can be studied for weeks. When returned to expansion conditions, the organoids readily resume their growth. We apply this condition to AOs established from nasal inferior turbinate brush samples of patients suffering from primary ciliary dyskinesia (PCD), a pulmonary disease caused by dysfunction of the motile cilia in the airways. Patient‐specific differences in ciliary beating are observed and are in agreement with the patients' genetic mutations. More detailed organoid ciliary phenotypes can thus be documented in addition to the standard diagnostic procedure. Additionally, using genetic editing tools, we show that a patient‐specific mutation can be repaired. This study demonstrates the utility of organoid technology for investigating hereditary airway diseases such as PCD.

Inhibiting Notch activity in breast cancer stem cells by functionalized gold nanoparticles with gamma-secretase inhibitor DAPT and vitamin C

Inhibiting Notch activity in breast cancer stem cells by functionalized gold nanoparticles with gamma-secretase inhibitor DAPT and vitamin C

Notch Signaling Pathway Is Activated by Sulfate Reducing Bacteria.

Sulfate Reducing Bacteria (SRB), usually rare residents of the gut, are often found in increased numbers (called a SRB bloom) in inflammatory conditions such as Inflammatory Bowel Disease (IBD), pouchitis, and periodontitis. However, the underlying mechanisms of this association remain largely unknown. Notch signaling, a conserved cell-cell communication pathway, is usually involved in tissue development and differentiation. Dysregulated Notch signaling is observed in inflammatory conditions such as IBD. Lipolysaccharide and pathogens also activate Notch pathway in macrophages. In this study, we tested whether Desulfovibrio, the most dominant SRB genus in the gut, may activate Notch signaling. RAW 264.7 macrophages were infected with Desulfovibrio vulgaris (DSV) and analyzed for the expression of Notch signaling pathway-related proteins. We found that DSV induced protein expression of Notch1 receptor, Notch intracellular domain (NICD) and p21, a downstream Notch target, in a dose-and time-dependent manner. DSV also induced the expression of pro-IL1β, a precursor of IL-1β, and SOCS3, a regulator of cytokine signaling. The gamma secretase inhibitor DAPT or Notch siRNA dampened DSV-induced Notch-related protein expression as well the expression of pro-IL1β and SOCS3. Induction of Notch-related proteins by DSV was not affected by TLR4 -IN -C34(C34), a TLR4 receptor antagonist. Additionally, cell-free supernatant of DSV-infected macrophages induced NICD expression in uninfected macrophages. DSV also activated Notch pathway in the human epithelial cell line HCT116 and in mouse small intestine. Thus, our study uncovers a novel mechanism by which SRB interact with host cells by activating Notch signaling pathway. Our study lays a framework for examining whether the Notch pathway induced by SRB contributes to inflammation in conditions associated with SRB bloom and whether it can be targeted as a therapeutic approach to treat these conditions.

Effects of the Gamma Secretase Inhibitors (GSI) Dapt (GSI-IX) and LSN424354 on Gene Expression and Shedding of Bcma from Multiple Myeloma and Chronic Lymphocytic Leukemia Tumor Cells

Introduction: Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) patients have high levels of solubilized (s) BCMA in the blood, and this protein is responsible for the immune deficiency of MM. sBCMA may also interfere with the efficacy of anti-BCMA-CAR-transduced T cells or other anti-BCMA antibody-based immunotherapy for multiple myeloma patients. Recently, γ-secretase has been identified as the enzyme that leads to the shedding of BCMA from B-cells. Gamma secretase (GS) is a multi-subunit protease complex that includes four individual proteins: presenilin-1 (PSEN1), nicastrin, anterior pharynx-defective 1 (APH-1), and presenilin enhancer 2 (PEN-2). CD147 acts as a non-essential regulator of the complex. We examined gene expression of PSEN1, APH-1, PEN-2, and CD147 among MM pts with progressive disease (PD) and complete remission (CR). We also determined the effects of the GS inhibitors (i) DAPT (GSI-IX) and LSN424354 (Eli Lilly, Indianapolis, IN) on solubilized BCMA levels from cultured human MM and CLL tumor cells.Study design: Bone marrow (BM) mononuclear cells (MCs) and serum were collected from monoclonal gammopathy of undetermined significance (MGUS) individuals, MM patients and healthy subjects after obtaining IRB approval. BMMCs from MM patients were treated with or without DAPT (GSI-IX) in a concentration dependent fashion for 24 hours. Following incubation, cells were washed three times using PBS. Flow cytometric analysis was performed using a Beckman Coulter Cytomics FC 500 with Cytomics CXP software. Total RNA was extracted using the Qiagen RNeasy kit (Qiagen, Louisville, KY) following the manufacturer's instructions. Quantitative PCR (qPCR) was applied to measure the relative abundance of human PSEN1, APH-1, PEN-2, and CD147 mRNA compared to that of the housekeeping gene HPRT mRNA. Single-cell suspensions were prepared from human MM LAGκ-1A xenografts which had been grown in the left superficial gluteal muscle of SCID mice. Cells were cultured and treated with the LSN424354 in a concentration dependent fashion. Supernatant BCMA levels were determined using an ELISA (R&D Systems, Minneapolis, MN) assay.Results and Discussion: qPCR showed PEN-2 gene expression was only slightly increased among MM patients with progressive disease (PD) compared to those in complete response or normal subjects whereas there was no change in expression of the PSEN1 or APH1 genes. CD147 gene expression was markedly increased in MM pts in PD (n=25) compared with those in CR (n=18; P=0.005) or MGUS (n=9; P=0.005). Next, we examined the effect of the DAPT (GSI-IX) on primary human MM tumor cells. Supernatants from primary human MM cells cultured for 24 hours in DAPT showed markedly reduced BCMA in a concentration dependent fashion (range,0.2 nM-2µM). Using qPCR, DAPT markedly reduced PEN-2 gene expression in a concentration dependent fashion. On the other hand, this GSI did not effect BCMA gene expression. DAPT reduces shedding of BCMA from B-cells but not its gene expression. We also determined BCMA levels in supernatants from cultured tumor cells derived from the human MM LAGκ-1A xenograft following 5 days of tissue culture with LSN424354 at concentrations ranging from 100 pM to 10 nM. Supernatant sBCMA levels were reduced > 90% at the highest concentrations (10nM). Similarly, freshly obtained CLL tumor cells cultured with LSN424354 at concentrations ranging from 100 pM to 10 nM also showed a marked reduction in supernatant BCMA levels at the higher concentrations.Conclusion: Gamma secretase sheds BCMA off B-cells. CD147, a regulator of gamma secretase activity, shows markedly higher gene expression among MM pts with PD compared with those in CR or MGUS individuals. The GSIs DAPT and LSN424354 reduce shedding of BCMA from malignant cells from MM and CLL patients. Since GSIs reduce solubilized BCMA levels, it is possible that treatment with these agents can reverse the immune dysfunction caused by this solubilized protein, and also improve the efficacy of anti-BCMA immunotherapies for treating patients with multiple myeloma and CLL. DisclosuresBerenson:Incyte: Consultancy, Research Funding; OncoTracker, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Long non-coding RNA HOTAIR induces GLI2 expression through Notch signalling in systemic sclerosis dermal fibroblasts

ObjectivesSystemic sclerosis (SSc) is characterised by tissue fibrosis of the major organs of the body including the skin, lungs and heart. We have previously reported that the lncRNA HOTAIR plays a central role in the activation of SSc myofibroblasts, the key cellular elements of fibrosis. HOTAIR induces fibroblast activation through H3K27me3-mediated activation of the Notch signalling pathway. Here we aimed to identify the signalling events downstream of Notch that drive SSc myofibroblast activation.MethodsPatient fibroblasts were obtained from full-thickness forearm skin biopsies of 3 adult patients with SSc of recent onset. The lncRNA HOTAIR was expressed in healthy dermal fibroblasts by lentiviral transduction. Hedgehog signalling pathway was inhibited with GANT61 and GLI2 siRNA. Gamma secretase inhibitors RO4929097 and DAPT were used to block Notch signalling. GSK126 was used to inhibit Enhancer of Zeste 2 (EZH2).ResultsOverexpression of HOTAIR in dermal fibroblasts induced the expression of the Hedgehog pathway transcription factor GLI2. This is mediated by activation of Notch signalling following epigenetic downregulation of miRNA-34a expression. Inhibition of H3K27 methylation and Notch signalling reduced expression of GLI2 in HOTAIR-expressing fibroblasts as well as in SSc dermal fibroblasts. Importantly, the inhibition of GLI2 function using GANT61 or siRNA mitigates the pro-fibrotic phenotype induced by HOTAIR.ConclusionsOur data indicates that GLI2 expression is stably upregulated in SSc myofibroblasts through HOTAIR and that GLI2 mediates the expression of pro-fibrotic markers downstream of Notch.

Organoid cultures of MELAS neural cells reveal hyperactive Notch signaling that impacts neurodevelopment

Mutations in mitochondrial DNA (mtDNA), typically maternally inherited, can result in severe neurological conditions. There is currently no cure for mitochondrial DNA diseases and treatments focus on management of the symptoms rather than correcting the defects downstream of the mtDNA mutation. Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) is one such mitochondrial disease that affects many bodily systems, particularly the central nervous system and skeletal muscles. Given the motor deficits seen in MELAS patients, we investigate the contribution of motor neuron pathology to MELAS. Using a spinal cord organoid system derived from induced pluripotent stem cells of a MELAS patient, as well as its isogenically corrected control, we found that high levels of Notch signaling underlie neurogenesis delays and neurite outgrowth defects that are associated with MELAS neural cultures. Furthermore, we demonstrate that the gamma-secretase inhibitor DAPT can reverse these neurodevelopmental defects.

Loss of Notch1 Activity Inhibits Prostate Cancer Growth and Metastasis and Sensitizes Prostate Cancer Cells to Antiandrogen Therapies.

Prostate cancer remains among the leading causes of cancer-related deaths in men. Patients with aggressive disease typically undergo hormone deprivation therapy. Although treatment is initially very successful, these men commonly progress to lethal, castration-resistant prostate cancer (CRPC) in 2 to 3 years. Standard therapies for CRPC include second-generation antiandrogens, which prolong patient lifespan by only several months. It is imperative to advance our understanding of the mechanisms leading to resistance to identify new therapies for aggressive prostate cancer. This study identifies Notch1 as a therapeutic target in prostate cancer. Loss of NOTCH1 in aggressive prostate cancer cells decreases proliferation, invasion, and tumorsphere formation. Therapeutic inhibition of Notch1 activity with gamma secretase inhibitors RO4929097 or DAPT in prostate cancer cells further results in decreased proliferative abilities. Loss of NOTCH1 and treatment of immunocompromised mice bearing prostate cancer xenografts with RO4929097 display significantly impaired tumor growth. Loss of NOTCH1 additionally decreased metastatic potential of prostate cancer cells in invasion assays in vitro as well as in vivo experiments. Moreover, treatment with gamma secretase inhibitors or NOTCH1 gene deletion synergized with antiandrogen therapies, enzalutamide or abiraterone, to decrease the growth of prostate cancer cells. Combination of gamma secretase inhibitors with abiraterone significantly inhibited cell migration and invasion, while combination with enzalutamide reversed enzalutamide-induced migration and invasion. These collective findings suggest loss of NOTCH1 delays growth of CRPC and inhibits metastasis, and inhibition of Notch1 activation in conjunction with second-generation antiandrogen therapies could delay growth and progression of prostate cancer.

HUMAN IPSC-DERIVED ALZHEIMER’S DISEASE ASTROCYTES RECAPITULATE DISEASE-RELATED PHENOTYPES

The importance of astrocytes in neurodegenerative disorders, including Alzheimer's disease (AD), is well recognized. Yet, their exact role in the disease pathogenesis and progression remains unclear. Interestingly, human astrocytes have been found to be far larger and more complex than those of infraprimate mammals, making it necessary to concentrate research on diseased human astrocytes rather than animal cells. In this study, patient-specific induced pluripotent stem cells (iPSCs) were used as a source for human astrocytes. iPSCs were reprogrammed from skin fibroblasts of familial AD patients with deletion of exon 9 in the PSEN1gene (n=3) and from healthy age-matched controls (n=3). The deletion of exon 9 was corrected with CRISPR/Cas9 genome editing, enabling generation of isogenic control lines for the diseased lines. iPSCs were then further differentiated to GFAP and S100b-positive forebrain astrocytes according to our optimized protocol. iPSC-derived astrocytes are functional cells that are able to propagate calcium waves, uptake glucose, produce cytokines and glutathione, and release gliotransmitters, such as GABA and ATP. In addition, AD astrocytes show calcium leakage from ER and reduced presenilin endoproteolysis, both well-characterized defects linked to PSEN1mutations. Interestingly, inflammatory stimulation of AD astrocytes resulted in increased release of proinflammatory cytokines when compared to control cells. AD astrocytes also have alterations in mitochondrial mass, cellular metabolism and glutathione levels. Most importantly, astrocytes contribute to amyloid pathology and are able to both produce and uptake beta-amyloid. AD astrocytes secrete significantly higher amounts of the 1–42 form and show an elevated 1–42/1–40 ratio as compared to controls, but respond well to treatment with gamma-secretase inhibitor DAPT. Our data show that pathological findings commonly seen in AD patients are recapitulated in the iPSC-derived astrocytes. Thus, these cells offer a valuable tool for studying the disease mechanisms and for drug screening and testing novel therapeutic approaches in a cell-type specific manner.

Combined EGFR- and notch inhibition display additive inhibitory effect on glioblastoma cell viability and glioblastoma-induced endothelial cell sprouting in vitro.

BackgroundFor Glioblastoma (GBM) patients, a number of anti-neoplastic strategies using specifically targeting drugs have been tested; however, the effects on survival have been limited. One explanation could be treatment resistance due to redundant signaling pathways, which substantiates the need for combination therapies. In GBM, both the epidermal growth factor receptor (EGFR) and the notch signaling pathways are often deregulated and linked to cellular growth, invasion and angiogenesis. Several studies have confirmed cross-talk and co-dependence of these pathways. Therefore, this study aimed at testing a combination treatment strategy using inhibitors targeting the notch and EGFR pathways.MethodsFor evaluation of cell viability a standard MTT assay was used. Western blotting (WB) and Q-RT-PCR were employed in order to assess the protein- and mRNA expression levels, respectively. In order to determine angiogenic processes, we used an endothelial spheroid sprouting assay. For assessment of secreted VEGF from GBM cells we performed a VEGF-quantikine ELISA.ResultsGBM cells were confirmed to express EGFR and Notch and to have the capacity to induce endothelial cell sprouting. Inhibition of EGFR and Notch signaling was achieved using either Iressa (gefitinib) or the gamma-secretase inhibitor DAPT. Our data showed that DAPT combined with Iressa treatment displayed increased inhibitory effect on cell viability and abrogated expression and activation of major pro-survival pathways. Similarly, the combinational treatment significantly increased abrogation of GBM-induced endothelial cell sprouting suggesting reduced GBM angiogenesis.ConclusionThis study finds that simultaneous targeting of notch and EGFR signaling leads to enhanced inhibitory effects on GBM-induced angiogenesis and cell viability, thereby stressing the importance of further evaluation of this targeting approach in a clinical setting.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-016-0309-2) contains supplementary material, which is available to authorized users.

Aβ-mediated spine changes in the hippocampus are microtubule-dependent and can be reversed by a subnanomolar concentration of the microtubule-stabilizing agent epothilone D

Dendritic spines represent the major postsynaptic input of excitatory synapses. Loss of spines and changes in their morphology correlate with cognitive impairment in Alzheimer's disease (AD) and are thought to occur early during pathology. Therapeutic intervention at a preclinical stage of AD to modify spine changes might thus be warranted. To follow the development and to potentially interfere with spine changes over time, we established a long term ex vivo model from organotypic cultures of the hippocampus from APP transgenic and control mice. The cultures exhibit spine loss in principal hippocampal neurons, which closely resembles the changes occurring in vivo, and spine morphology progressively changes from mushroom-shaped to stubby. We demonstrate that spine changes are completely reversed within few days after blocking amyloid-β (Aβ) production with the gamma-secretase inhibitor DAPT. We show that the microtubule disrupting drug nocodazole leads to spine loss similar to Aβ expressing cultures and suppresses DAPT-mediated spine recovery in slices from APP transgenic mice. Finally, we report that epothilone D (EpoD) at a subnanomolar concentration, which slightly stabilizes microtubules in model neurons, completely reverses Aβ-induced spine loss and increases thin spine density. Taken together the data indicate that Aβ causes spine changes by microtubule destabilization and that spine recovery requires microtubule polymerization. Moreover, our results suggest that a low, subtoxic concentration of EpoD is sufficient to reduce spine loss during the preclinical stage of AD.

Tissue regeneration and biomineralization in sea urchins: role of Notch signaling and presence of stem cell markers.

Echinoderms represent a phylum with exceptional regenerative capabilities that can reconstruct both external appendages and internal organs. Mechanistic understanding of the cellular pathways involved in regeneration in these animals has been hampered by the limited genomic tools and limited ability to manipulate regenerative processes. We present a functional assay to investigate mechanisms of tissue regeneration and biomineralization by measuring the regrowth of amputated tube feet (sensory and motor appendages) and spines in the sea urchin, Lytechinus variegatus. The ability to manipulate regeneration was demonstrated by concentration-dependent inhibition of regrowth of spines and tube feet by treatment with the mitotic inhibitor, vincristine. Treatment with the gamma-secretase inhibitor DAPT resulted in a concentration-dependent inhibition of regrowth, indicating that both tube feet and spine regeneration require functional Notch signaling. Stem cell markers (Piwi and Vasa) were expressed in tube feet and spine tissue, and Vasa-positive cells were localized throughout the epidermis of tube feet by immunohistochemistry, suggesting the existence of multipotent progenitor cells in these highly regenerative appendages. The presence of Vasa protein in other somatic tissues (e.g. esophagus, radial nerve, and a sub-population of coelomocytes) suggests that multipotent cells are present throughout adult sea urchins and may contribute to normal homeostasis in addition to regeneration. Mechanistic insight into the cellular pathways governing the tremendous regenerative capacity of echinoderms may reveal processes that can be modulated for regenerative therapies, shed light on the evolution of regeneration, and enable the ability to predict how these processes will respond to changing environmental conditions.

Abstract 1369: Inhibition of Notch- and EGFR signaling reduces cell viability and angiogenesis in glioblastoma multiforme

Abstract The high-grade glioma, Glioblastoma Multiforme (GBM), is the most prevalent and aggressive brain tumour in adults with a median survival of fifteen months. Thus new therapeutic targets are urgently needed. GBM is characterized by extensive vascularisation why targeting angiogenesis has promising potential in improving the treatment of GBM patients. Aberrant EGFR signaling is another characteristic of GBM, and increased EGFR downstream signaling is believed to correlate with poor prognosis. However, little is known of the involvement of EGFR signaling in GBM angiogenesis. Notch signaling is known for its role in angiogenesis during foetal development and in other cancers, but the importance of Notch signaling in GBM angiogenesis is yet to be elucidated. Recently, it has been shown, that crosstalk between Notch- and EGFR signaling occurs in cancer cells, and aberrant EGFR signaling may therefore play a functional role in GBM angiogenesis through crosstalk with Notch signaling. The aim of this study was to investigate the effect of combined Notch- and EGFR inhibition on GBM cell viability and on ability of GBM cells to induce angiogenesis in endothelial cells. In vitro assays of GBM angiogenesis were established using two patient derived GBM cell cultures, one expressing the mutated EGFR, the EGFRvIII, and one expressing wild type EGFR. The functional relevance of EGFR- and Notch signaling in GBM cells was tested using the tyrosine kinase inhibitor Iressa for EGFR inhibition, and the gamma-secretase inhibitor DAPT for Notch inhibition. We show that combined inhibition of EGFR- and Notch signaling caused additive inhibition of cell viability of both cell cultures in vitro. Abrogated EGFR/Notch signaling in GBM cells further reduced angiogenesis in endothelial cells exposed to conditioned media from treated GBM cells. Moreover, when GBM cells were treated with the inhibitors, this inhibited phosphorylation and activation of the two survival signalling pathways ERK and Akt, and abrogated secretion and expression of the most prominent angiogenic factor - VEGF. The presented results indicate that EGFR- and Notch signaling play a functional role in GBM angiogenesis and that combined treatment with drugs targeting both the EGFR- and Notch pathway could prove beneficial over single drug therapy in targeting both the tumorigenic and angiogenic potential of GBM cells. Citation Format: Mikkel Staberg, Signe Regner Michaelsen, Louise Stobbe Olsen, Mette Kjølhede Nedergaard, Mette Villingshøj, Hans Skovgaard Poulsen. Inhibition of Notch- and EGFR signaling reduces cell viability and angiogenesis in glioblastoma multiforme. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1369. doi:10.1158/1538-7445.AM2015-1369

Improving Pancreatic Islet Engraftment after Islet Transplantation through Administration of Gamma-Secretase Inhibitor DAPT

Rapid and effective revascularization of transplanted pancreatic islets is vital for the survival and function of the islet graft. Insufficient vascularization after islet transplantation may be one causative factor to the failure of islet grafts in clinical transplantation. The aim of this study was to investigate if N-{N-(2-(3,5-Difluorophenyl)acetyl)-(S)-alanyl}- (S)-phenylglycine- tert-butyl ester (DAPT) administration can improve engraftment of transplanted islets. DAPT is a dipeptidic gamma-secretase inhibitor which inhibits Notch signaling. Notch signaling is involved in angiogenesis and inhibition may result in excessive formation of new blood vessels. Excessive vasculature may be beneficial in the immediate posttransplantation period since the transplanted islets are dependent on diffusion of oxygen and nutrients before revascularization. Islets isolated from C57BL/6 mice were transplanted beneath the renal capsule of C57BL/6 mice. After islet transplantation DAPT or vehicle was administered subcutaneously for three days. Mice treated with DAPT had an increased vascular density when compared to control mice two days and one month posttransplantation. Moreover, mice treated with DAPT showed 54±8.2 % functional blood vessels compared to 40±6.7 % in control mice two days posttransplantation. After one month, the fraction of functional blood vessels increased to 86±2.8 % in DAPT treated mice compared to 61±9.4 % in control mice. Our findings demonstrated that administration of DAPT may be a feasible strategy to improve engraftment of transplanted islets.

Inhibition of Notch signaling alters the phenotype of orthotopic tumors formed from glioblastoma multiforme neurosphere cells but does not hamper intracranial tumor growth regardless of endogene Notch pathway signature

Background Brain cancer stem-like cells (bCSC) are cancer cells with neural stem cell (NSC)-like properties found in the devastating brain tumor glioblastoma multiforme (GBM). bCSC are proposed a central role in tumor initiation, progression, treatment resistance and relapse and as such present a promising target in GBM research. The Notch signaling pathway is often deregulated in GBM and we have previously characterized GBM-derived bCSC cultures based on their expression of the Notch-1 receptor and found that it could be used as predictive marker for the effect of Notch inhibition. The aim of the present project was therefore to further elucidate the significance of Notch pathway activity for the tumorigenic properties of GBM-derived bCSC.Methods Human-derived GBM xenograft cells previously established as NSC-like neurosphere cultures were used. Notch inhibition was accomplished by exposing the cells to the gamma-secretase inhibitor DAPT prior to gene expression analysis and intracranial injection into immunocompromised mice.Results By analyzing the expression of several Notch pathway components, we found that the cultures indeed displayed different Notch pathway signatures. However, when DAPT-treated neurosphere cells were injected into the brain of immunocompromised mice, no increase in survival was obtained regardless of Notch pathway signature and Notch inhibition. We did however observe a decrease in the expression of the stem cell marker Nestin, an increase in the proliferative marker Ki-67 and an increased number of abnormal vessels in tumors formed from DAPT-treated, high Notch-1 expressing cultures, when compared with the control.Conclusion Based on the presented results we propose that Notch inhibition partly induces differentiation of bCSC, and selects for a cell type that more strongly induces angiogenesis if the treatment is not sustained. However, this more differentiated cell type might prove to be more sensitive to conventional therapies.

Neuronal apoptosis and synaptic density in the dentate gyrus of ischemic rats' response to chronic mild stress and the effects of Notch signaling.

Our previous research highlighted an inconsistency with Notch1 signaling-related compensatory neurogenesis after chronic mild stress (CMS) in rodents suffering from cerebral ischemia, which continue to display post-stroke depressive symptoms. Here, we hypothesize that CMS aggrandized ischemia-related apoptosis injury and worsened synaptic integrity via gamma secretase-meditated Notch1 signaling. Adult rats were exposed to a CMS paradigm after left middle cerebral artery occlusion (MCAO). Open-field and sucrose consumption testing were employed to assess depression-like behavior. Gene expression of pro-apoptotic Bax, anti-apoptotic Bcl-2, and synaptic density-related synaptophysin were measured by western blotting and real-time PCR on Day 28 after MCAO surgery. CMS induced depressive behaviors in ischemic rats, which was accompanied by an elevation in Bax/bcl-2 ratio, TUNEL staining in neurons and reduced synaptophysin expression in the dentate gyrus. These collective effects were reversed by the gamma-secretase inhibitor DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenyl-glycine t-butyl ester). We found that post-stroke stressors made neurons in the dentate gyrus vulnerable to apoptosis, which supports a putative role for Notch signaling in neural integrity, potentially in newborn cells’ synaptic deficit with regard to preexisting cells. These findings suggest that post-stroke depression therapeutically benefits from blocking gamma secretase mediated Notch signaling, and whether this signaling pathway could be a therapeutic target needs to be further investigated.