A γ-secretase inhibitor blocks Notch signaling in vivo and causes a severe neurogenic phenotype in zebrafish

Zebrafish Neurogenetics Junior Research Group, Institute for Virology, Technical University Munich, Trogerstrasse, Germany.
EMBO Reports (Impact Factor: 9.06). 08/2002; 3(7):688-94. DOI: 10.1093/embo-reports/kvf124
Source: PubMed


Inhibition of amyloid beta-peptide (Abeta) production by blocking gamma-secretase activity is at present one of the most promising therapeutic strategies to slow progression of Alzheimer's disease pathology. gamma-secretase inhibitors apparently block Abeta generation via interference with presenilin (PS) function. Besides being an essential component of the gamma-secretase complex, PS itself may be an aspartyl protease with gamma-secretase activity, which is not only required for Abeta production but also for a similar proteolytic process involved in Notch signaling. Here we demonstrate that treatment of zebrafish embryos with a known gamma-secretase inhibitor affects embryonic development in a manner indistinguishable from Notch signaling deficiencies at morphological, molecular and biochemical levels. This indicates severe side-effects of gamma-secretase inhibitors in any Notch-dependent cell fate decision and demonstrates that the zebrafish is an ideal vertebrate system to validate compounds that selectively affect Abeta production, but not Notch signaling, under in vivo conditions.


Available from: Michael Willem
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    • "divergent signalling pathways or as is the case for Notch, allows for the translocation of ICDs to the nucleus where they enable transcriptional activation activity [22]. Indeed, Psen1 knockout animals display a predominant Notch loss-of function phenotype resulting from loss of γ-secretase cleavage of Notch, highlighting the importance of γ-secretase in Notch signalling [23] [24] [25]. Cleavage can also be used as a signal for degradation of transmembrane protein fragments and the maintenance of so-called 'membrane proteostasis' [26]. "
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    ABSTRACT: The presenilins are the catalytic subunit of the membrane-embedded tetrameric γ-secretase protease complexes. More that 90 transmembrane proteins have been reported to be γ-secretase substrates, including the widely studied amyloid precursor protein (APP) and the Notch receptor, which are precursors for the generation of amyloid-β peptides and biologically active APP intracellular domain (AICD) and Notch intracellular domain (NICD). The diversity of γ-secretase substrates highlights the importance of presenilin-dependent γ-secretase protease activities as a regulatory mechanism in a range of biological systems. However, there is also a growing body of evidence that supports the existence of γ-secretase-independent functions for the presenilins in the regulation and progression of an array of cell signalling pathways. In this review, we will present an overview of current literature that proposes evolutionarily conserved presenilin functions outside of the γ-secretase complex, with a focus on the suggested role of the presenilins in the regulation of Wnt/β-catenin signalling, protein trafficking and degradation, calcium homeostasis and apoptosis.
    Cellular Signalling 10/2015; 28(1). DOI:10.1016/j.cellsig.2015.10.006 · 4.32 Impact Factor
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    • "We confirmed the role of Notch signaling in the miR-132 morphant phenotype using the well characterized g-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylgly- cine t-butyl ester (DAPT) (Geling et al., 2002) to inhibit Notch from 24 hpf on, thus covering the period when the Notch pathway has been shown to actively instruct the radial glial cell fate and arborization (Kim et al., 2008b). DAPT represses her4 transcription in the zebrafish spinal cord (Kim et al., 2008b). "
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    ABSTRACT: Radial glial progenitors play pivotal roles in the development and patterning of the spinal cord, and their fate is controlled by Notch signaling. How Notch is shaped to regulate their crucial transition from expansion toward differentiation remains, however, unknown. miR-132 in the developing zebrafish dampens Notch signaling via a cascade involving the transcriptional corepressor Ctbp2 and the Notch suppressor Sirt1. At early embryonic stages, high Ctbp2 levels sustain Notch signaling and radial glial expansion and concomitantly induce miR-132 expression via a double-negative feedback loop involving Rest inhibition. The changing balance in miR-132 and Ctbp2 interaction gradually drives the switch in Notch output and radial glial progenitor fate as part of the larger developmental program involved in the transition from embryonic to larval spinal cord.
    Developmental Cell 08/2014; 30(4). DOI:10.1016/j.devcel.2014.07.006 · 9.71 Impact Factor
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    • "To confirm that prostate cells have active Notch signaling [33], RWPE1 cells, derived from benign prostate epithelium, and PC-3 prostate cancer cells were treated with the γ-secretase inhibitor DAPT, known to prevent Notch processing and transcriptional signaling [48]. This treatment caused a significant downregulation of the endogenous Notch target genes HES1 and HEY1, as determined by real-time RT-PCR (Figure 1A) and a comparable decline in the HES1 promoter activity, as determined by luciferase transactivation assays (Figure 1A). "
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    ABSTRACT: PTOV1 is an adaptor protein with functions in diverse processes, including gene transcription and protein translation, whose overexpression is associated with a higher proliferation index and tumor grade in prostate cancer (PC) and other neoplasms. Here we report its interaction with the Notch pathway and its involvement in PC progression. Stable PTOV1 knockdown or overexpression were performed by lentiviral transduction. Protein interactions were analyzed by co-immunoprecipitation, pull-down and/or immunofluorescence. Endogenous gene expression was analyzed by real time RT-PCR and/or Western blotting. Exogenous promoter activities were studied by luciferase assays. Gene promoter interactions were analyzed by chromatin immunoprecipitation assays (ChIP). In vivo studies were performed in the Drosophila melanogaster wing, the SCID-Beige mouse model, and human prostate cancer tissues and metastasis. The Excel package was used for statistical analysis. Knockdown of PTOV1 in prostate epithelial cells and HaCaT skin keratinocytes caused the upregulation, and overexpression of PTOV1 the downregulation, of the Notch target genes HEY1 and HES1, suggesting that PTOV1 counteracts Notch signaling. Under conditions of inactive Notch signaling, endogenous PTOV1 associated with the HEY1 and HES1 promoters, together with components of the Notch repressor complex. Conversely, expression of active Notch1 provoked the dismissal of PTOV1 from these promoters. The antagonist role of PTOV1 on Notch activity was corroborated in the Drosophila melanogaster wing, where human PTOV1 exacerbated Notch deletion mutant phenotypes and suppressed the effects of constitutively active Notch. PTOV1 was required for optimal in vitro invasiveness and anchorage-independent growth of PC-3 cells, activities counteracted by Notch, and for their efficient growth and metastatic spread in vivo. In prostate tumors, the overexpression of PTOV1 was associated with decreased expression of HEY1 and HES1, and this correlation was significant in metastatic lesions. High levels of the adaptor protein PTOV1 counteract the transcriptional activity of Notch. Our evidences link the pro-oncogenic and pro-metastatic effects of PTOV1 in prostate cancer to its inhibitory activity on Notch signaling and are supportive of a tumor suppressor role of Notch in prostate cancer progression.
    Molecular Cancer 03/2014; 13(1):74. DOI:10.1186/1476-4598-13-74 · 4.26 Impact Factor
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