Discovery of senolytics using machine learning.

Cellular senescence is a stress response involved in ageing and diverse disease processes including cancer, type-2 diabetes, osteoarthritis and viral infection. Despite growing interest in targeted elimination of senescent cells, only few senolytics are known due to the lack of well-characterised molecular targets. Here, we report the discovery of three senolytics using cost-effective machine learning algorithms trained solely on published data. We computationally screened various chemical libraries and validated the senolytic action of ginkgetin, periplocin and oleandrin in human cell lines under various modalities of senescence. The compounds have potency comparable to known senolytics, and we show that oleandrin has improved potency over its target as compared to best-in-class alternatives. Our approach led to several hundred-fold reduction in drug screening costs and demonstrates that artificial intelligence can take maximum advantage of small and heterogeneous drug screening data, paving the way for new open science approaches to early-stage drug discovery.

Failure of digit tip regeneration in the absence of Lmx1b suggests Lmx1b functions disparate from dorsoventral polarity.

Mammalian digit tip regeneration is linked to the presence of nail tissue, but a nail-explicit model is missing. Here, we report that nail-less double-ventral digits of ΔLARM1/2 mutants that lack limb-specific Lmx1b enhancers fail to regenerate. To separate the nail's effect from the lack of dorsoventral (DV) polarity, we also interrogate double-dorsal double-nail digits and show that they regenerate. Thus, DV polarity is not a prerequisite for regeneration, and the nail requirement is supported. Transcriptomic comparison between wild-type and non-regenerative ΔLARM1/2 mutant blastemas reveals differential upregulation of vascularization and connective tissue functional signatures in wild type versus upregulation of inflammation in the mutant. These results, together with the finding of Lmx1b expression in the postnatal dorsal dermis underneath the nail and uniformly in the regenerative blastema, open the possibility of additional Lmx1b roles in digit tip regeneration, in addition to the indirect effect of mediating the formation of the nail.

Structural connectivity and subcellular changes after antidepressant doses of ketamine and Ro 25-6981 in the rat: an MRI and immuno-labeling study.

Ketamine has rapid and robust antidepressant effects. However, unwanted psychotomimetic effects limit its widespread use. Hence, several studies examined whether GluN2B-subunit selective NMDA antagonists would exhibit a better therapeutic profile. Although preclinical work has revealed some of the mechanisms of action of ketamine at cellular and molecular levels, the impact on brain circuitry is poorly understood. Several neuroimaging studies have examined the functional changes in the brain induced by acute administration of ketamine and Ro 25-6981 (a GluN2B-subunit selective antagonist), but the changes in the microstructure of gray and white matter have received less attention. Here, the effects of ketamine and Ro 25-6981 on gray and white matter integrity in male Sprague-Dawley rats were determined using diffusion-weighted magnetic resonance imaging (DWI). In addition, DWI-based structural brain networks were estimated and connectivity metrics were computed at the regional level. Immunohistochemical analyses were also performed to determine whether changes in myelin basic protein (MBP) and neurofilament heavy-chain protein (NF200) may underlie connectivity changes. In general, ketamine and Ro 25-6981 showed some opposite structural alterations, but both compounds coincided only in increasing the fractional anisotropy in infralimbic prefrontal cortex and dorsal raphe nucleus. These changes were associated with increments of NF200 in deep layers of the infralimbic cortex (together with increased MBP) and the dorsal raphe nucleus. Our results suggest that the synthesis of NF200 and MBP may contribute to the formation of new dendritic spines and myelination, respectively. We also suggest that the increase of fractional anisotropy of the infralimbic and dorsal raphe nucleus areas could represent a biomarker of a rapid antidepressant response.

An intrinsic timer specifies distal structures of the vertebrate limb.

How the positional values along the proximo-distal axis (stylopod-zeugopod-autopod) of the limb are specified is intensely debated. Early work suggested that cells intrinsically change their proximo-distal positional values by measuring time. Recently, however, it is suggested that instructive extrinsic signals from the trunk and apical ectodermal ridge specify the stylopod and zeugopod/autopod, respectively. Here, we show that the zeugopod and autopod are specified by an intrinsic timing mechanism. By grafting green fluorescent protein-expressing cells from early to late chick wing buds, we demonstrate that distal mesenchyme cells intrinsically time Hoxa13 expression, cell cycle parameters and the duration of the overlying apical ectodermal ridge. In addition, we reveal that cell affinities intrinsically change in the distal mesenchyme, which we suggest results in a gradient of positional values along the proximo-distal axis. We propose a complete model in which a switch from extrinsic signalling to intrinsic timing patterns the vertebrate limb.

Endocytosis as a biological response in receptor pharmacology: evaluation by fluorescence microscopy.

The activation of G-protein coupled receptors by agonist compounds results in diverse biological responses in cells, such as the endocytosis process consisting in the translocation of receptors from the plasma membrane to the cytoplasm within internalizing vesicles or endosomes. In order to functionally evaluate endocytosis events resulted from pharmacological responses, we have developed an image analysis method -the Q-Endosomes algorithm- that specifically discriminates the fluorescent signal originated at endosomes from that one observed at the plasma membrane in images obtained from living cells by fluorescence microscopy. Mu opioid (MOP) receptor tagged at the carboxy-terminus with yellow fluorescent protein (YFP) and permanently expressed in HEK293 cells was used as experimental model to validate this methodology. Time-course experiments performed with several agonists resulted in different sigmoid curves depending on the drug used to initiate MOP receptor endocytosis. Thus, endocytosis resulting from the simultaneous activation of co-expressed MOP and serotonin 5-HT2C receptors by morphine plus serotonin was significantly different, in kinetics as well as in maximal response parameters, from the one caused by DAMGO, sufentanyl or methadone. Therefore, this analytical tool permits the pharmacological characterization of receptor endocytosis in living cells with functional and temporal resolution.

A screen for transcription factor targets of glycogen synthase kinase-3 highlights an inverse correlation of NFκB and androgen receptor signaling in prostate cancer.

Expression of Glycogen Synthase Kinase-3 (GSK-3) is elevated in prostate cancer and its inhibition reduces prostate cancer cell proliferation, in part by reducing androgen receptor (AR) signaling. However, GSK-3 inhibition can also activate signals that promote cell proliferation and survival, which may preclude the use of GSK-3 inhibitors in the clinic. To identify such signals in prostate cancer, we screened for changes in transcription factor target DNA binding activity in GSK-3-silenced cells. Among the alterations was a reduction in AR DNA target binding, as predicted from previous studies, and an increase in NFκB DNA target binding. Consistent with the latter, gene silencing of GSK-3 or inhibition using the GSK-3 inhibitor CHIR99021 increased basal NFκB transcriptional activity. Activation of NFκB was accompanied by an increase in the level of the NFκB family member RelB. Conversely, silencing RelB reduced activation of NFκB by CHIR99021. Furthermore, the reduction of prostate cancer cell proliferation by CHIR99021 was potentiated by inhibition of NFκB signaling using the IKK inhibitor PS1145. Finally, stratification of human prostate tumor gene expression data for GSK3 revealed an inverse correlation between NFκB-dependent and androgen-dependent gene expression, consistent with the results from the transcription factor target DNA binding screen. In addition, there was a correlation between expression of androgen-repressed NFκB target genes and reduced survival of patients with metastatic prostate cancer. These findings highlight an association between GSK-3/AR and NFκB signaling and its potential clinical importance in metastatic prostate cancer.

Distinct expression and activity of GSK-3α and GSK-3ß in prostate cancer.

Glycogen synthase kinase (GSK-3) is upregulated in many types of tumor, including prostate cancer. GSK-3 inhibitors reduce prostate tumor cell growth; however, it is not clear if both isoforms, GSK-3α and GSK-3ß, are involved. Here, we compared their expression in prostate tumors and used gene silencing to study their functions in 22Rv1 prostate cancer cells. Compared to normal prostate, GSK-3α and GSK-3ß were upregulated in 25/79 and 24/79 cases of prostate cancer, respectively, with GSK-3α elevated in low Gleason sum score tumors and GSK-3ß expressed in high Gleason tumors, and both isoforms correlating with high expression of the androgen receptor (AR). Gene silencing of GSK-3α and, to a lesser extent, GSK-3ß reduced AR transcriptional activity. In addition, silencing of GSK-3ß, but not GSK-3α, reduced Akt phosphorylation. Acute and chronic silencing of either isoform reduced 22Rv1 growth in colony formation assays; however, this did not correlate with effects on AR activity. The GSK-3 inhibitor CHIR99021 reduced 22Rv1 colony formation by 50% in normal growth medium and by 15% in hormone-depleted medium, suggesting that GSK-3 is required both for hormone-dependent and hormone-independent proliferation. In addition, CHIR99021 enhanced growth inhibition by the AR antagonists bicalutamide and MDV3100. Finally, expression of GSK3A and GSK3B mRNAs correlated with a gene expression signature for androgen-regulated genes. Our observations highlight the importance of the GSK-3/AR signaling axis in prostate cancer and support the case for development of isoform-specific GSK-3 inhibitors and their use, in combination with AR antagonists, to treat patients with prostate cancer.

Distinct roles for Wnt-4 and Wnt-11 during retinoic acid-induced neuronal differentiation.

Retinoic acid and Wnt/ß-catenin signals play important roles during neuronal differentiation but less is known about noncanonical Wnt signals in this context. We examined retinoic acid and Wnt signaling in two human embryonal carcinoma cell lines, NTERA-2 (clone D1), which undergoes neuronal differentiation in response to retinoic acid, and 2102Ep, which does not. Retinoic acid treatment inhibited ß-catenin/Tcf activity in NTERA-2 cells but not in 2102Ep cells. Inhibition occurred downstream of ß-catenin but did not involve competition between retinoic acid receptors and ß-catenin for binding to p300 or Tcf-4. Ectopic expression of FZD1 partially restored inhibition in 2102Ep cells, suggesting the involvement of Wnt ligands. Retinoic acid treatment of NTERA-2 cells induced the expression of Wnt-4 and Wnt-11, both of which were able to inhibit ß-catenin/Tcf activity. Wnt-4 and Wnt-11 were found at cell borders in islands of cells that expressed OCT4 and GFAP and were predominantly negative for Nestin, PAX6, and GATA6. Gene silencing of Wnt-4, but not Wnt-11, reduced retinoic acid downregulation of OCT4 and Nanog and upregulation of PAX6, ASCL1, HOXC5, and NEUROD1, suggesting that Wnt-4 promotes early neuronal differentiation. Gene expression analysis of NTERA-2 cells stably overexpressing Wnt-11 suggested that Wnt-11 potentiates retinoic acid induction of early neurogenesis. Consistent with this, overexpression of Wnt-11 maintained a population of proliferating progenitor cells in cultures treated with retinoic acid for several weeks. These observations highlight the distinct roles of two noncanonical Wnts during the early stages of retinoic acid-induced neuronal differentiation.

Issues associated with the use of phosphospecific antibodies to localise active and inactive pools of GSK-3 in cells.

BACKGROUND: Glycogen synthase kinase-3 (GSK-3) is a ubiquitously expressed serine/threonine (Ser/Thr) kinase comprising two isoforms, GSK-3α and GSK-3ß. Both enzymes are similarly inactivated by serine phosphorylation (GSK-3α at Ser21 and GSK-3ß at Ser9) and activated by tyrosine phosphorylation (GSK-3α at Tyr279 and GSK-3ß at Tyr216). Antibodies raised to phosphopeptides containing the sequences around these phosphorylation sites are frequently used to provide an indication of the activation state of GSK-3 in cell and tissue extracts. These antibodies have further been used to determine the subcellular localisation of active and inactive forms of GSK-3, and the results of those studies support roles for GSK-3 phosphorylation in diverse cellular processes. However, the specificity of these antibodies in immunocytochemistry has not been addressed in any detail. RESULTS: Taking advantage of gene silencing technology, we examined the specificity of several commercially available anti-phosphorylated GSK-3 antibodies. We show that antibodies raised to peptides containing the phosphorylated Ser21/9 epitope crossreact with unidentified antigens that are highly expressed by mitotic cells and that mainly localise to spindle poles. In addition, two antibodies raised to peptides containing the phosphorylated Tyr279/216 epitope recognise an unidentified protein at focal contacts, and a third antibody recognises a protein found in Ki-67-positive cell nuclei. While the phosphorylated Ser9/21 GSK-3 antibodies also recognise other proteins whose levels increase in mitotic cells in western blots, the phosphorylated Tyr279/216 antibodies appear to be specific in western blotting. However, we cannot rule out the posssibility that they recognise very large or very small proteins that might not be detected using a standard western blotting approach. CONCLUSIONS: Our findings indicate that care should be taken when examining the subcellular localisation of active or inactive GSK-3 and, furthermore, suggest that the role of GSK-3 phosphorylation in some cellular processes be reassessed.

Notch activates cell cycle reentry and progression in quiescent cardiomyocytes.

The inability of heart muscle to regenerate by replication of existing cardiomyocytes has engendered considerable interest in identifying developmental or other stimuli capable of sustaining the proliferative capacity of immature cardiomyocytes or stimulating division of postmitotic cardiomyocytes. Here, we demonstrate that reactivation of Notch signaling causes embryonic stem cell-derived and neonatal ventricular cardiomyocytes to enter the cell cycle. The proliferative response of neonatal ventricular cardiomyocytes declines as they mature, such that late activation of Notch triggers the DNA damage checkpoint and G2/M interphase arrest. Notch induces recombination signal-binding protein 1 for Jkappa (RBP-Jkappa)-dependent expression of cyclin D1 but, unlike other inducers, also shifts its subcellular distribution from the cytosol to the nucleus. Nuclear localization of cyclin D1 is independent of RBP-Jkappa. Thus, the influence of Notch on nucleocytoplasmic localization of cyclin D1 is an unanticipated property of the Notch intracellular domain that is likely to regulate the cell cycle in multiple contexts, including tumorigenesis as well as cardiogenesis.

TNF triggers mitogenic signals in NIH 3T3 cells but induces apoptosis when the cell cycle is blocked.

Tumor necrosis factor (TNF) is known to be a mediator of a variety of cellular responses including apoptotic death or proliferation depending on the target cell and the environmental conditions. We show here that TNF triggers both growth and death signals in NIH 3T3 murine fibroblasts. In cells arrested in G(0) by serum deprivation, TNF drives approximately 50% of them to enter the cell cycle, but kills the cells that remain quiescent. The presence of serum prevents toxic effects of TNF, suggesting that TNF can cooperate to drive cells through the cell cycle, but is unable to do so by itself and alternatively it triggers death signals in cells unable to proliferate. Interestingly, TNF induces a similar toxic effect in cells forced to stay at the G(1)/S border, S or M phases. We have explored the TNF apoptotic pathway in arrested cells. This mechanism is not due to the loss of the anti-apoptotic capacity of NFkappaB and is mediated by mitochondria since Bcl-2 overexpression partially inhibits cell death. There are, however, interesting differences in the kinetics of mitochondrial events which indicate that this form of sensitization to TNF leads to an apoptotic mechanism different from that observed after sensitization by RNA synthesis inhibition.

Isolation and characterization of nudC from mouse macrophages, a gene implicated in the inflammatory response through the regulation of PAF-AH(I) activity.

We report the characterization of a cDNA induced in mouse macrophages that encodes a 332-amino acid protein with extensive sequence identity with members of the mammalian nudC-like genes. The interaction between mNUDC and the regulatory beta subunit of platelet activating factor acetylhydrolase I (PAF-AH(I)) shown in this article indicates a new function of NUDC. Thus, we show that NUDC increases the catalytic activity of PAF-AH(I) and that this regulatory activity is located in the carboxyl terminal half of the protein which is highly conserved. This suggests a novel function for mammalian nudC-like genes as anti-inflammatory proteins.