Cellular Senescence: Defining a Path Forward.

Cellular senescence is a cell state implicated in various physiological processes and a wide spectrum of age-related diseases. Recently, interest in therapeutically targeting senescence to improve healthy aging and age-related disease, otherwise known as senotherapy, has been growing rapidly. Thus, the accurate detection of senescent cells, especially in vivo, is essential. Here, we present a consensus from the International Cell Senescence Association (ICSA), defining and discussing key cellular and molecular features of senescence and offering recommendations on how to use them as biomarkers. We also present a resource tool to facilitate the identification of genes linked with senescence, SeneQuest (available at http://Senequest.net). Lastly, we propose an algorithm to accurately assess and quantify senescence, both in cultured cells and in vivo.

Age-related changes in the local milieu of inflamed tissues cause aberrant neutrophil trafficking and subsequent remote organ damage.

Aging is associated with dysregulated immune functions. Here, we investigated the impact of age on neutrophil diapedesis. Using confocal intravital microscopy, we found that in aged mice, neutrophils adhered to vascular endothelium in inflamed tissues but exhibited a high frequency of reverse transendothelial migration (rTEM). This retrograde breaching of the endothelium by neutrophils was governed by enhanced production of the chemokine CXCL1 from mast cells that localized at endothelial cell (EC) junctions. Increased EC expression of the atypical chemokine receptor 1 (ACKR1) supported this pro-inflammatory milieu in aged venules. Accumulation of CXCL1 caused desensitization of the chemokine receptor CXCR2 on neutrophils and loss of neutrophil directional motility within EC junctions. Fluorescent tracking revealed that in aged mice, neutrophils undergoing rTEM re-entered the circulation and disseminated to the lungs where they caused vascular leakage. Thus, neutrophils stemming from a local inflammatory site contribute to remote organ damage, with implication to the dysregulated systemic inflammation associated with aging.

Senescent endothelial cells promote pathogenic neutrophil trafficking in inflamed tissues.

Cellular senescence is a hallmark of advanced age and a major instigator of numerous inflammatory pathologies. While endothelial cell (EC) senescence is aligned with defective vascular functionality, its impact on fundamental inflammatory responses in vivo at single-cell level remain unclear. To directly investigate the role of EC senescence on dynamics of neutrophil-venular wall interactions, we applied high resolution confocal intravital microscopy to inflamed tissues of an EC-specific progeroid mouse model, characterized by profound indicators of EC senescence. Progerin-expressing ECs supported prolonged neutrophil adhesion and crawling in a cell autonomous manner that additionally mediated neutrophil-dependent microvascular leakage. Transcriptomic and immunofluorescence analysis of inflamed tissues identified elevated levels of EC CXCL1 on progerin-expressing ECs and functional blockade of CXCL1 suppressed the dysregulated neutrophil responses elicited by senescent ECs. Similarly, cultured progerin-expressing human ECs exhibited a senescent phenotype, were pro-inflammatory and prompted increased neutrophil attachment and activation. Collectively, our findings support the concept that senescent ECs drive excessive inflammation and provide new insights into the mode, dynamics, and mechanisms of this response at single-cell level.

Yearning for machine learning: applications for the classification and characterisation of senescence.

Senescence is a widely appreciated tumour suppressive mechanism, which acts as a barrier to cancer development by arresting cell cycle progression in response to harmful stimuli. However, senescent cell accumulation becomes deleterious in aging and contributes to a wide range of age-related pathologies. Furthermore, senescence has beneficial roles and is associated with a growing list of normal physiological processes including wound healing and embryonic development. Therefore, the biological role of senescent cells has become increasingly nuanced and complex. The emergence of sophisticated, next-generation profiling technologies, such as single-cell RNA sequencing, has accelerated our understanding of the heterogeneity of senescence, with distinct final cell states emerging within models as well as between cell types and tissues. In order to explore data sets of increasing size and complexity, the senescence field has begun to employ machine learning (ML) methodologies to probe these intricacies. Most notably, ML has been used to aid the classification of cells as senescent, as well as to characterise the final senescence phenotypes. Here, we provide a background to the principles of ML tasks, as well as some of the most commonly used methodologies from both traditional and deep ML. We focus on the application of these within the context of senescence research, by addressing the utility of ML for the analysis of data from different laboratory technologies (microscopy, transcriptomics, proteomics, methylomics), as well as the potential within senolytic drug discovery. Together, we aim to highlight both the progress and potential for the application of ML within senescence research.

Remodelling of microRNAs in colorectal cancer by hypoxia alters metabolism profiles and 5-fluorouracil resistance.

Solid tumours have oxygen gradients and areas of near and almost total anoxia. Hypoxia reduces sensitivity to 5-fluorouracil (5-FU)-chemotherapy for colorectal cancer (CRC). MicroRNAs (miRNAs) are hypoxia sensors and were altered consistently in six CRC cell lines (colon cancer: DLD-1, HCT116 and HT29; rectal cancer: HT55, SW837 and VACO4S) maintained in hypoxia (1 and 0.2% oxygen) compared with normoxia (20.9%). CRC cell lines also showed altered amino acid metabolism in hypoxia and hypoxia-responsive miRNAs were predicted to target genes in four metabolism pathways: beta-alanine; valine, leucine, iso-leucine; aminoacyl-tRNA; and alanine, aspartate, glutamate. MiR-210 was increased in hypoxic areas of CRC tissues and hypoxia-responsive miR-21 and miR-30d, but not miR-210, were significantly increased in 5-FU resistant CRCs. Treatment with miR-21 and miR-30d antagonists sensitized hypoxic CRC cells to 5-FU. Our data highlight the complexity and tumour heterogeneity caused by hypoxia. MiR-210 as a hypoxic biomarker, and the targeting of miR-21 and miR-30d and/or the amino acid metabolism pathways may offer translational opportunities.

Primary cilium-dependent and -independent Hedgehog signaling inhibits p16(INK4A).

In a genome-wide siRNA analysis of p16(INK4a) (p16) modulators, we identify the Hedgehog (Hh) pathway component SUFU and formally demonstrate that Hh signaling promotes mitogenesis by suppression of p16. A fragment of the Hh-responsive GLI2 transcription factor directly binds and inhibits the p16 promoter and senescence is associated with the loss of nuclear GLI2. Hh components partially reside in the primary cilium (PC), and the small fraction of cells in mass culture that elaborate a PC have the lowest expression of p16. Suppression of p16 is effected by both PC-dependent and -independent routes, and ablation of p16 renders cells insensitive to an Hh inhibitor and increases PC formation. These results directly link a well-established developmental mitogenic pathway with a key tumor suppressor and contribute to the molecular understanding of replicative senescence, Hh-mediated oncogenesis, and potentially the role of p16 in aging.

MCL-1 is modulated in Crohn's disease fibrosis by miR-29b via IL-6 and IL-8.

The miR-29 family is involved in fibrosis in multiple organs, including the intestine where miR-29b facilitates TGF-ß-mediated up-regulation of collagen in mucosal fibroblasts from Crohn's disease (CD) patients. Myeloid cell leukemia-1 (MCL-1), a member of the B-cell CLL/Lymphoma 2 (BCL-2) apoptosis family, is involved in liver fibrosis and is targeted by miR-29b via its 3'-UTR in cultured cell lines. We investigate the role of MCL-1 and miR-29b in primary intestinal fibroblasts and tissue from stricturing CD patients. Transfection of CD intestinal fibroblasts with pre-miR-29b resulted in a significant increase in the mRNA expression of MCL-1 isoforms [MCL-1Long (L)/Extra Short (ES) and MCL-1Short (S)], although MCL-1S was expressed at significantly lower levels. Western blotting predominantly detected the anti-apoptotic MCL-1L isoform, and immunofluorescence showed that staining was localised in discrete nuclear foci. Transfection with pre-miR-29b or anti-miR-29b resulted in a significant increase or decrease, respectively, in MCL-1L foci. CD fibroblasts treated with IL-6 and IL-8, inflammatory cytokines upstream of MCL-1, increased the total mass of MCL-1L-positive foci. Furthermore, transfection of intestinal fibroblasts with pre-miR-29b resulted in an increase in mRNA and protein levels of IL-6 and IL-8. Finally, immunohistochemistry showed reduced MCL-1 protein expression in fibrotic CD samples compared to non-stricturing controls. Together, our findings suggest that induction of MCL-1 by IL-6/IL-8 may surmount any direct down-regulation by miR-29b via its 3'-UTR. We propose that an anti-fibrotic miR-29b/IL-6 IL-8/MCL-1L axis may influence intestinal fibrosis in CD. In the future, therapeutic modulation of this pathway might contribute to the management of fibrosis in CD.

Brief report: isogenic induced pluripotent stem cell lines from an adult with mosaic down syndrome model accelerated neuronal ageing and neurodegeneration.

Trisomy 21 (T21), Down Syndrome (DS) is the most common genetic cause of dementia and intellectual disability. Modeling DS is beginning to yield pharmaceutical therapeutic interventions for amelioration of intellectual disability, which are currently being tested in clinical trials. DS is also a unique genetic system for investigation of pathological and protective mechanisms for accelerated ageing, neurodegeneration, dementia, cancer, and other important common diseases. New drugs could be identified and disease mechanisms better understood by establishment of well-controlled cell model systems. We have developed a first nonintegration-reprogrammed isogenic human induced pluripotent stem cell (iPSC) model of DS by reprogramming the skin fibroblasts from an adult individual with constitutional mosaicism for DS and separately cloning multiple isogenic T21 and euploid (D21) iPSC lines. Our model shows a very low number of reprogramming rearrangements as assessed by a high-resolution whole genome CGH-array hybridization, and it reproduces several cellular pathologies seen in primary human DS cells, as assessed by automated high-content microscopic analysis. Early differentiation shows an imbalance of the lineage-specific stem/progenitor cell compartments: T21 causes slower proliferation of neural and faster expansion of hematopoietic lineage. T21 iPSC-derived neurons show increased production of amyloid peptide-containing material, a decrease in mitochondrial membrane potential, and an increased number and abnormal appearance of mitochondria. Finally, T21-derived neurons show significantly higher number of DNA double-strand breaks than isogenic D21 controls. Our fully isogenic system therefore opens possibilities for modeling mechanisms of developmental, accelerated ageing, and neurodegenerative pathologies caused by T21.

Low Stress Ion Conductance Microscopy of Sub-Cellular Stiffness.

Directly examining subcellular mechanics whilst avoiding excessive strain of a live cell requires the precise control of light stress on very small areas, which is fundamentally difficult. Here we use a glass nanopipet out of contact with the plasma membrane to both exert the stress on the cell and also accurately monitor cellular compression. This allows the mapping of cell stiffness at a lateral resolution finer than 100 nm. We calculate the stress a nanopipet exerts on a cell as the sum of the intrinsic pressure between the tip face and the plasma membrane plus its direct pressure on any glycocalyx, both evaluated from the gap size in terms of the ion current decrease. A survey of cell types confirms that an intracellular pressure of approximately 120 Pa begins to detach the plasma membrane from the cytoskeleton and reveals that the first 0.66 ± 0.09 µm of compression of a neuron cell body is much softer than previous methods have been able to detect.

Cellular senescence mediated by p16INK4A-coupled miRNA pathways.

p16 is a key regulator of cellular senescence, yet the drivers of this stable state of proliferative arrest are not well understood. Here, we identify 22 senescence-associated microRNAs (SA-miRNAs) in normal human mammary epithelial cells. We show that SA-miRNAs-26b, 181a, 210 and 424 function in concert to directly repress expression of Polycomb group (PcG) proteins CBX7, embryonic ectoderm development (EED), enhancer of zeste homologue 2 (EZH2) and suppressor of zeste 12 homologue (Suz12), thereby activating p16. We demonstrate the existence of a tight positive feedback loop in which SA-miRNAs activate and re-enforce the expression of other SA-miRNA members. In contrast, PcG members restrain senescence by epigenetically repressing the expression of these SA-miRNAs. Importantly, loss of p16 leads to repression of SA-miRNA expression, intimately coupling this effector of senescence to the SA-miRNA/PcG self-regulatory loop. Taken together, our findings illuminate an important regulatory axis that underpins the transition from proliferation to cellular senescence.

In Crohn's disease fibrosis-reduced expression of the miR-29 family enhances collagen expression in intestinal fibroblasts.

Intestinal fibrosis with stricture formation is a complication of CD (Crohn's disease) that may mandate surgical resection. Accurate biomarkers that reflect the relative contribution of fibrosis to an individual stricture are an unmet need in managing patients with CD. The miRNA-29 (miR-29) family has been implicated in cardiac, hepatic and pulmonary fibrosis. In the present study, we investigated the expression of miR-29a, miR-29b and miR-29c in mucosa overlying a stricture in CD patients (SCD) paired with mucosa from non-strictured areas (NSCD). There was significant down-regulation of the miR-29 family in mucosa overlying SCD compared with mucosa overlying NSCD. miR-29b showed the largest fold-decrease and was selected for functional analysis. Overexpression of miR-29b in CD fibroblasts led to a down-regulation of collagen I and III transcripts and collagen III protein, but did not alter MMP (matrix metalloproteinase)-3, MMP-12 and TIMP (tissue inhibitor of metalloproteinase)-1 production. TGF (transforming growth factor)-ß1 up-regulated collagen I and III transcripts and collagen III protein as a consequence of the down-regulation of miR-29b, and TGF-ß1-induced collagen expression was reversed by exogenous overexpression of miR-29b. Furthermore, serum levels of miR-29 were lower in patients with stricturing disease compared with those without. These findings implicate the miR-29 family in the pathogenesis of intestinal fibrosis in CD and provide impetus for the further evaluation of the miR-29 family as biomarkers.

An expedited screening platform for the discovery of anti-ageing compounds in vitro and in vivo.

BACKGROUND: Restraining or slowing ageing hallmarks at the cellular level have been proposed as a route to increased organismal lifespan and healthspan. Consequently, there is great interest in anti-ageing drug discovery. However, this currently requires laborious and lengthy longevity analysis. Here, we present a novel screening readout for the expedited discovery of compounds that restrain ageing of cell populations in vitro and enable extension of in vivo lifespan. METHODS: Using Illumina methylation arrays, we monitored DNA methylation changes accompanying long-term passaging of adult primary human cells in culture. This enabled us to develop, test, and validate the CellPopAge Clock, an epigenetic clock with underlying algorithm, unique among existing epigenetic clocks for its design to detect anti-ageing compounds in vitro. Additionally, we measured markers of senescence and performed longevity experiments in vivo in Drosophila, to further validate our approach to discover novel anti-ageing compounds. Finally, we bench mark our epigenetic clock with other available epigenetic clocks to consolidate its usefulness and specialisation for primary cells in culture. RESULTS: We developed a novel epigenetic clock, the CellPopAge Clock, to accurately monitor the age of a population of adult human primary cells. We find that the CellPopAge Clock can detect decelerated passage-based ageing of human primary cells treated with rapamycin or trametinib, well-established longevity drugs. We then utilise the CellPopAge Clock as a screening tool for the identification of compounds which decelerate ageing of cell populations, uncovering novel anti-ageing drugs, torin2 and dactolisib (BEZ-235). We demonstrate that delayed epigenetic ageing in human primary cells treated with anti-ageing compounds is accompanied by a reduction in senescence and ageing biomarkers. Finally, we extend our screening platform in vivo by taking advantage of a specially formulated holidic medium for increased drug bioavailability in Drosophila. We show that the novel anti-ageing drugs, torin2 and dactolisib (BEZ-235), increase longevity in vivo. CONCLUSIONS: Our method expands the scope of CpG methylation profiling to accurately and rapidly detecting anti-ageing potential of drugs using human cells in vitro, and in vivo, providing a novel accelerated discovery platform to test sought after anti-ageing compounds and geroprotectors.

Death-seq and ye shall find: A novel screening strategy for dying cells.

Killing senescent cells to improve health-span holds great promise. However, screening for senescence-regulating genes and molecules is challenging because these cells do not proliferate. In this issue, Colville and Liu et al. develop Death-seq, a positive selection screening tool that overcomes this hurdle to offer broad genetic and pharmacological utility.

Phytochemistry and Cytotoxic Activity of Aquilaria crassna Pericarp on MDA-MB-468 Cell Lines.

The extracts of Aquilaria crassna pericarp were investigated on the MDA-MB-468, a breast cancer cell line, at desired concentration (1-50 µg/mL). The results showed that the dichloromethane (DCM) extract exhibited the strongest toxicity and was carried out subsequently. A total of nine compounds were isolated from the DCM extract using column chromatography and recrystallization, of which their structures were determined. Intriguingly, in addition to the previously reported compounds, neocucurbitacin A, a cucurbitacin triterpenoid aglycone with a lactone in ring A, was reported for the first time in the Aquilaria genus. Among the isolated compounds, cucurbitacin E highly inhibited MDA-MB-468 cell growth in a dose-dependent manner. Owing to binding abilities with the SH2 domain in the molecular docking study, cucurbitacin E, neocucurbitan A, neocucurbitan B, and cucurbitacin E 2-O-ß-d-glucopyranoside act as STAT3 inhibitors and are suitable for further research. This study suggests thatAquilaria crassnafruits could serve as a promising source of natural compounds with potential anticancer effects, particularly against breast cancer.

Current Understanding of the Role of Senescent Melanocytes in Skin Ageing.

Melanocytes reside within the basal epidermis of human skin, and function to protect the skin from ultraviolet light through the production of melanin. Prolonged exposure of the skin to UV light can induce irreparable DNA damage and drive cells into senescence, a sustained cell cycle arrest that prevents the propagation of this damage. Senescent cells can also be detrimental and contribute to skin ageing phenotypes through their senescence-associated secretory phenotype. Senescent cells can act in both an autocrine and paracrine manner to produce widespread tissue inflammation and skin ageing. Recently, melanocytes have been identified as the main senescent cell population within the epidermis and have been linked to a variety of skin ageing phenotypes, such as epidermal thinning and the presence of wrinkles. However, the literature surrounding melanocyte senescence is limited and tends to focus on the role of senescence in the prevention of melanoma. Therefore, this review aims to explore the current understanding of the contribution of senescent melanocytes to human skin ageing.

Senescence-associated morphological profiles (SAMPs): an image-based phenotypic profiling method for evaluating the inter and intra model heterogeneity of senescence.

Senescence occurs in response to a number of damaging stimuli to limit oncogenic transformation and cancer development. As no single, universal senescence marker has been discovered, the confident classification of senescence induction requires the parallel assessment of a series of hallmarks. Therefore, there is a growing need for "first-pass" tools of senescence identification to streamline experimental workflows and complement conventional markers. Here, we utilise a high content, multidimensional phenotypic profiling-based approach, to assess the morphological profiles of senescent cells induced via a range of stimuli. In the context of senescence, we refer to these as senescence-associated morphological profiles (SAMPs), as they facilitate distinction between senescent and proliferating cells. The complexity of the profiles generated also allows exploration of the heterogeneity both between models of senescence and within an individual senescence model, providing a level of insight at the single cell level. Furthermore, we also demonstrate that these models are applicable to the assessment of senescence in vivo, which remains a key challenge for the field. Therefore, we believe SAMPs has the potential to serve as a useful addition in the repertoire of senescence researchers, either as a first-pass tool or as part of the established senescence hallmarks.

A whole genome screen for HIV restriction factors.

BACKGROUND: Upon cellular entry retroviruses must avoid innate restriction factors produced by the host cell. For human immunodeficiency virus (HIV) human restriction factors, APOBEC3 (apolipoprotein-B-mRNA-editing-enzyme), p21 and tetherin are well characterised. RESULTS: To identify intrinsic resistance factors to HIV-1 replication we screened 19,121 human genes and identified 114 factors with significant inhibition of infection. Those with a known function are involved in a broad spectrum of cellular processes including receptor signalling, vesicle trafficking, transcription, apoptosis, cross-nuclear membrane transport, meiosis, DNA damage repair, ubiquitination and RNA processing. We focused on the PAF1 complex which has been previously implicated in gene transcription, cell cycle control and mRNA surveillance. Knockdown of all members of the PAF1 family of proteins enhanced HIV-1 reverse transcription and integration of provirus. Over-expression of PAF1 in host cells renders them refractory to HIV-1. Simian Immunodeficiency Viruses and HIV-2 are also restricted in PAF1 expressing cells. PAF1 is expressed in primary monocytes, macrophages and T-lymphocytes and we demonstrate strong activity in MonoMac1, a monocyte cell line. CONCLUSIONS: We propose that the PAF1c establishes an anti-viral state to prevent infection by incoming retroviruses. This previously unrecognised mechanism of restriction could have implications for invasion of cells by any pathogen.

Isolation methodology is essential to the evaluation of the extracellular vesicle component of the senescence-associated secretory phenotype.

A hallmark of senescence is the acquisition of an enhanced secretome comprising inflammatory mediators and tissue remodelling agents - the senescence-associated secretory phenotype (SASP). Through the SASP, senescent cells are hypothesised to contribute to both ageing and pathologies associated with age. Whilst soluble factors have been the most widely investigated components of the SASP, there is growing evidence that small extracellular vesicles (EVs) comprise functionally important constituents. Thus, dissecting the contribution of the soluble SASP from the vesicular component is crucial to elucidating the functional significance of senescent cell derived EVs. Here, we take advantage of a systematic proteomics based approach to determine that soluble SASP factors co-isolate with EVs following differential ultracentrifugation (dUC). We present size-exclusion chromatography (SEC) as a method for separation of the soluble and vesicular components of the senescent secretome and thus EV purification. Furthermore, we demonstrate that SEC EVs isolated from senescent cells contribute to non-cell autonomous paracrine senescence. Therefore, this work emphasises the requirement for methodological rigor due to the propensity of SASP components to co-isolate during dUC and provides a framework for future investigations of the vesicular component of the SASP.

Expression of p16 Within Myenteric Neurons of the Aged Colon: A Potential Marker of Declining Function.

Human colonic neuromuscular functions decline among the elderly. The aim was to explore the involvement of senescence. A preliminary PCR study looked for age-dependent differences in expression of CDKN1A (encoding the senescence-related p21 protein) and CDKN2A (encoding p16 and p14) in human ascending and descending colon (without mucosa) from 39 (approximately 50: 50 male: female) adult (aged 27-60 years) and elderly donors (70-89 years). Other genes from different aging pathways (e.g., inflammation, oxidative stress, autophagy) and cell-types (e.g., neurons, neuron axonal transport) were also examined. Unlike CDKN1A, CDKN2A (using primers for p16 and p14 but not when using p14-specific primers) was upregulated in both regions of colon. Compared with the number of genes appearing to upregulate in association with temporal age, more genes positively associated with increased CDKN2A expression (respectively, 16 and five of 44 genes studied for ascending and descending colon). Confirmation of increased expression of CDKN2A was sought by immunostaining for p16 in the myenteric plexus of colon from 52 patients, using a semi-automated software protocol. The results showed increased staining not within the glial cells (S100 stained), but in the cytoplasm of myenteric nerve cell bodies (MAP2 stained, with identified nucleus) of ascending, but not descending colon of the elderly, and not in the cell nucleus of either region or age group (5,710 neurons analyzed: n = 12-14 for each group). It was concluded that increased p16 staining within the cytoplasm of myenteric nerve cell bodies of elderly ascending (but not descending) colon, suggests a region-dependent, post-mitotic cellular senescence-like activity, perhaps involved with aging of enteric neurons within the colon.

Early growth response 2 (EGR2) is a novel regulator of the senescence programme.

Senescence, a state of stable growth arrest, plays an important role in ageing and age-related diseases in vivo. Although the INK4/ARF locus is known to be essential for senescence programmes, the key regulators driving p16 and ARF transcription remain largely underexplored. Using siRNA screening for modulators of the p16/pRB and ARF/p53/p21 pathways in deeply senescent human mammary epithelial cells (DS HMECs) and fibroblasts (DS HMFs), we identified EGR2 as a novel regulator of senescence. EGR2 expression is up-regulated during senescence, and its ablation by siRNA in DS HMECs and HMFs transiently reverses the senescent phenotype. We demonstrate that EGR2 activates the ARF and p16 promoters and directly binds to both the ARF and p16 promoters. Loss of EGR2 down-regulates p16 levels and increases the pool of p16- p21- 'reversed' cells in the population. Moreover, EGR2 overexpression is sufficient to induce senescence. Our data suggest that EGR2 is a direct transcriptional activator of the p16/pRB and ARF/p53/p21 pathways in senescence and a novel marker of senescence.