Selective Irreversible Inhibitors of the Wnt-Deacylating Enzyme NOTUM Developed by Activity-Based Protein Profiling

Suciu, Radu M.; Cognetta, Armand B.; Potter, Zachary E.; Cravatt, Benjamin F.

doi:10.1021/acsmedchemlett.8b00191

Cited by 41 publications

(55 citation statements)

References 31 publications

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“…7e-g). Finally, to test if the regenerative capacity of old intestines can be promoted via the intestinal stem cell niche, we used a novel small molecule inhibitor of Notum ABC99 31 . ABC99 blunted the effects of exogenous Notum and increased the frequency of Lgr5 hi cells in vitro (Extended Data Fig.…”

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confidence: 99%

“…Rapamycin treatment was performed as described previously12 . ABC99 was produced as described previously31 . 33,3 mg/ml stock solution in ethanol was prepared freshly and further mixed 1:1:1:17 into Tween-80 (Sigma), PEG-400 (HamiltonResearch) and 0,9% NaCl respectively.…”

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confidence: 99%

“…daily having the last dose 2h before sacrifice together with 10 mg/kg of EdU. Control mice were treated with vehicle or equal amount of inactive control compound ABC10131 . 5-Fluorouracil (Sigma) was reconstituted in DMSO 100 mg/ml and single i.p.…”

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Notum produced by Paneth cells attenuates regeneration of aged intestinal epithelium

Pentinmikko

Iqbal

Mana

et al. 2019

Nature

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A decline in stem cell function impairs tissue regeneration during aging, but the role of the stem cell supporting niche in aging is not well understood. The small intestine is maintained by actively cycling intestinal stem cells (ISCs) that are regulated by the Paneth cell niche 1,2. Here we show that the regenerative potential of human and mouse intestinal epithelium diminishes with age due to defects in both stem cells and their niche. The functional decline was caused by decrease in stemness maintaining Wnt signalling due to production of an extracellular Wnt-inhibitor, Notum, in aged Paneth cells. Mechanistically, high mTORC1 activity in old Paneth cells inhibits PPARa activity 3 and lowered PPARa increased Notum expression. Genetic targeting of Notum or Wnt-supplementation restored function of old intestinal organoids. Moreover, pharmacological inhibition of Notum in mice enhanced the regenerative capacity of old stem cells and promoted recovery from chemotherapy induced damage. Our results reveal an unappreciated role for the stem cell niche in aging and demonstrate that targeting of Notum can promote regeneration of old tissues. Tissue turnover and regenerative capacity decrease upon aging in many tissue types 4-6. The intestinal epithelium is one of the fastest renewing tissues in the human body and has been reported to regenerate without loss of self-renewal in long term in vitro organoid culture 7. However,

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Notum produced by Paneth cells attenuates regeneration of aged intestinal epithelium

Pentinmikko

Iqbal

Mana

et al. 2019

Nature

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203

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“…Given the context-dependent roles of WNT signaling in the V-SVZ ( Adachi et al, 2007 ; Azim et al, 2014 ; Ortega et al, 2013 ; Qu et al, 2010 ; Zywitza et al, 2018 ), we next examined the function of NOTUM in V-SVZ regulation. We modulated NOTUM activity in the V-SVZ using either a selective small molecule inhibitor (ABC99) ( Suciu et al, 2018 ) or a recombinant mouse NOTUM protein. We first investigated the effects of these perturbations ex vivo in organotypic slice cultures from 7- to 8-week-old Nestin - CFP reporter mice ( Mignone et al, 2004 ; Wojcinski et al, 2017 ) treated with vehicle (saline), recombinant mouse NOTUM protein, ABC99, or the inactive control compound ABC101 ( Figure 4A ).…”

Section: Resultsmentioning

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Single-Cell Profiling and SCOPE-Seq Reveal Lineage Dynamics of Adult Ventricular-Subventricular Zone Neurogenesis and NOTUM as a Key Regulator

et al. 2020

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SUMMARY In the adult ventricular-subventricular zone (V-SVZ), neural stem cells (NSCs) generate new olfactory bulb (OB) neurons and glia throughout life. To map adult neuronal lineage progression, we profiled >56,000 V-SVZ and OB cells by single-cell RNA sequencing (scRNA-seq). Our analyses reveal the molecular diversity of OB neurons, including fate-mapped neurons, lineage progression dynamics, and an NSC intermediate enriched for Notum , which encodes a secreted WNT antagonist. SCOPE-seq technology, which links live-cell imaging with scRNA-seq, uncovers cell-size transitions during NSC differentiation and preferential NOTUM binding to proliferating neuronal precursors. Consistently, application of NOTUM protein in slice cultures and pharmacological inhibition of NOTUM in slice cultures and in vivo demonstrated that NOTUM negatively regulates V-SVZ proliferation. Timely, context-dependent neurogenesis demands adaptive signaling among neighboring progenitors. Our findings highlight a critical regulatory state during NSC activation marked by NOTUM, which attenuates WNT-stimulated proliferation in NSC progeny.

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“…Given these findings and the crucial context-dependent roles of WNT signaling in orchestrating proliferation and differentiation in the V-SVZ (Adachi et al, 2007; Azim et al, 2014; Ortega et al, 2013; Qu et al, 2010; Zywitza et al, 2018), we hypothesized that NOTUM plays a critical role in regulating V-SVZ neuronal progenitors. To examine NOTUM function in the V-SVZ, we blocked NOTUM activity using a selective small molecule inhibitor (ABC99) (Suciu et al, 2018). To investigate effects of NOTUM inhibition ex vivo on the V-SVZ, we generated organotypic slice cultures from Nestin-CFP reporter mice (Mignone et al, 2004; Wojcinski et al, 2017), and treated them with ABC99 or the inactive control compound ABC101 ( Figure 4A ).…”

Section: Resultsmentioning

confidence: 99%

Single-cell profiling and SCOPE-seq reveal the lineage dynamics of adult neurogenesis and NOTUM as a key V-SVZ regulator

Mizrak

Bayın

Yuan

et al. 2019

Preprint

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24Neural stem cells (NSCs) and their progeny reside in specialized niches in the adult mammalian 25 brain where they generate new neurons and glia throughout life. Adult NSCs of the ventricular-26 subventricular zone (V-SVZ) are prone to rapid exhaustion; thus timely, context-dependent 27 neurogenesis demands adaptive signaling among the vast number of neighboring progenitors 28 nestled between the ventricular surface and nearby blood vessels. To dissect adult neuronal 29 lineage progression and regulation, we profiled >56,000 V-SVZ and olfactory bulb (OB) cells by 30 single-cell RNA-sequencing (scRNA-seq). Our analyses revealed the diversity of V-SVZ-derived 31 OB neurons, the temporal dynamics of lineage progression, and a key intermediate NSC 32 population enriched for expression of Notum, which encodes a secreted WNT antagonist. Single 33 Cell Optical Phenotyping and Expression (SCOPE-seq), a technology linking live cell imaging with 34 2 scRNA-seq, uncovered dynamic control of cell size concomitant with NSC differentiation with35 Notum + NSCs at a critical size poised for cell division, and a preference of NOTUM surface binding 36 to neuronal precursors with active WNT signaling. Finally, in vivo pharmacological inhibition of 37 NOTUM significantly expanded neuronal precursor pools in the V-SVZ. Our findings highlight a 38 critical regulatory state during NSC activation marked by NOTUM, a secreted enzyme that 39 ensures efficient neurogenesis by preventing WNT signaling activation in NSC progeny. 40 41 INTRODUCTION 42Neurogenesis persists in two principal regions of the adult mouse brain: the subgranular zone of 43 the dentate gyrus and the V-SVZ located in the walls of the lateral ventricles (Doetsch et al., 1999; 44 Fiorelli et al., 2015; Mirzadeh et al., 2008). The V-SVZ contains both quiescent (qNSCs) and 45 actively dividing NSCs (aNSCs) (Codega et al., 2014; Mich et al., 2014) with intrinsic regional 46 identities defined during embryogenesis (Fuentealba et al., 2015), generating different OB 47 interneuron subtypes or glia depending on their location (Brill et al., 2009; Delgado and Lim, 2015; 48 Kohwi et al., 2005; Lopez-Juarez et al., 2013; Merkle et al., 2014; Merkle et al., 2007; Mizrak et 49 al., 2019; Ortega et al., 2013; Zweifel et al., 2018). V-SVZ qNSCs are specialized astrocytes with 50 radial morphology (reviewed in Kriegstein and Alvarez-Buylla, 2009; Mirzadeh et al., 2008), and 51 once activated divide symmetrically, resulting in their depletion over time (Obernier et al., 2018). 52 aNSCs give rise to transit-amplifying cells (TACs), which generate neuroblasts (NBs). NBs 53 migrate via the rostral migratory stream (RMS) to the OB (Figure 1A), where they terminally 54 differentiate into interneurons. V-SVZ NSC-to-OB interneuron differentiation requires cells with a 55 broad distribution of developmental states; however, the identities and the functions of key 56 intermediate states and the gene expression programs pertinent to these transitions are unknown. 57 While recent effort...

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Selective Irreversible Inhibitors of the Wnt-Deacylating Enzyme NOTUM Developed by Activity-Based Protein Profiling

Cited by 41 publications

References 31 publications

Notum produced by Paneth cells attenuates regeneration of aged intestinal epithelium

Notum produced by Paneth cells attenuates regeneration of aged intestinal epithelium

Single-Cell Profiling and SCOPE-Seq Reveal Lineage Dynamics of Adult Ventricular-Subventricular Zone Neurogenesis and NOTUM as a Key Regulator

Single-cell profiling and SCOPE-seq reveal the lineage dynamics of adult neurogenesis and NOTUM as a key V-SVZ regulator

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