2'-O-ribose methylation levels of ribosomal RNA distinguish different types of growth arrest in human dermal fibroblasts.

The 2'-O-methylation (2'-O-Me) of ribosomal RNA (rRNA) shows plasticity that is potentially associated with cell phenotypes. We used RiboMeth-seq profiling to reveal growth arrest-specific 2'-O-Me patterns in primary human dermal fibroblasts from three different donors. We exposed cells to hydrogen peroxide to induce cellular senescence and to high cell densities to promote quiescence by contact inhibition. We compared both modes of cell cycle arrest to proliferating cells and could indeed distinguish these conditions by their overall 2'-O-Me patterns. Methylation levels at a small fraction of sites showed plasticity and correlated with the expression of specific small nucleolar RNAs (snoRNAs) but not with expression of fibrillarin. Moreover, we observed subtle senescence-associated alterations in ribosome biogenesis. Knockdown of the snoRNA SNORD87, which acts as a guide for modification of a hypermethylated position in non-proliferating cells, was sufficient to boost cell proliferation. Conversely, depletion of SNORD88A, SNORD88B and SNORD88C, which act as guides for modification of a hypomethylated site, caused decreased proliferation without affecting global protein synthesis or apoptosis. Taken together, our findings provide evidence that rRNA modifications can be used to distinguish and potentially influence specific growth phenotypes of primary cells.

Human diamine oxidase cellular binding and internalization in vitro and rapid clearance in vivo are not mediated by N-glycans but by heparan sulfate proteoglycan interactions.

Human diamine oxidase (hDAO) rapidly inactivates histamine by deamination. No pharmacokinetic data are available to better understand its potential as a new therapeutic modality for diseases with excess local and systemic histamine, like anaphylaxis, urticaria or mastocytosis. After intravenous administration of recombinant hDAO to rats and mice, more than 90% of the dose disappeared from the plasma pool within 10 min. Human DAO did not only bind to various endothelial and epithelial cell lines in vitro, but was also unexpectedly internalized and visible in granule-like structures. The uptake of rhDAO into cells was dependent on neither the asialoglycoprotein-receptor (ASGP-R) nor the mannose receptor (MR) recognizing terminal galactose or mannose residues, respectively. Competition experiments with ASGP-R and MR ligands did not block internalization in vitro or rapid clearance in vivo. The lack of involvement of N-glycans was confirmed by testing various glycosylation mutants. High but not low molecular weight heparin strongly reduced the internalization of rhDAO in HepG2 cells and HUVECs. Human DAO was readily internalized by CHO-K1 cells, but not by the glycosaminoglycan- and heparan sulfate-deficient CHO cell lines pgsA-745 and pgsD-677, respectively. A docked heparin hexasaccharide interacted well with the predicted heparin binding site 568RFKRKLPK575. These results strongly imply that rhDAO clearance in vivo and cellular uptake in vitro is independent of N-glycan interactions with the classical clearance receptors ASGP-R and MR, but is mediated by binding to heparan sulfate proteoglycans followed by internalization via an unknown receptor.

Loss of the ribosomal RNA methyltransferase NSUN5 impairs global protein synthesis and normal growth.

Modifications of ribosomal RNA expand the nucleotide repertoire and thereby contribute to ribosome heterogeneity and translational regulation of gene expression. One particular m5C modification of 25S ribosomal RNA, which is introduced by Rcm1p, was previously shown to modulate stress responses and lifespan in yeast and other small organisms. Here, we report that NSUN5 is the functional orthologue of Rcm1p, introducing m5C3782 into human and m5C3438 into mouse 28S ribosomal RNA. Haploinsufficiency of the NSUN5 gene in fibroblasts from William Beuren syndrome patients causes partial loss of this modification. The N-terminal domain of NSUN5 is required for targeting to nucleoli, while two evolutionary highly conserved cysteines mediate catalysis. Phenotypic consequences of NSUN5 deficiency in mammalian cells include decreased proliferation and size, which can be attributed to a reduction in total protein synthesis by altered ribosomes. Strikingly, Nsun5 knockout in mice causes decreased body weight and lean mass without alterations in food intake, as well as a trend towards reduced protein synthesis in several tissues. Together, our findings emphasize the importance of single RNA modifications for ribosome function and normal cellular and organismal physiology.

Modulation of mammalian translation by a ribosome-associated tRNA half.

Originally considered futile degradation products, tRNA-derived RNA fragments (tdRs) have been shown over the recent past to be crucial players in orchestrating various cellular functions. Unlike other small non-coding RNA (ncRNA) classes, tdRs possess a multifaceted functional repertoire ranging from regulating transcription, apoptosis, RNA interference, ribosome biogenesis to controlling translation efficiency. A subset of the latter tdRs has been shown to directly target the ribosome, the central molecular machine of protein biosynthesis. Here we describe the function of the mammalian tRNAPro 5' half, a 35 residue long ncRNA associated with ribosomes and polysomes in several mammalian cell lines. Addition of tRNAPro halves to mammalian in vitro translation systems results in global translation inhibition and concomitantly causes the upregulation of a specific low molecular weight translational product. This tRNAPro 5' half-dependent translation product consists of both RNA and amino acids. Transfection of the tRNAPro half into HeLa cells leads to the formation of the same product in vivo. The migration of this product in acidic gels, the insensitivity to copper sulphate treatment, the resistance to 3' polyadenylation, and the association with 80S monosomes indicate that the accumulated product is peptidyl-tRNA. Our data thus suggest that binding of the tRNAPro 5' half to the ribosome leads to ribosome stalling and to the formation of peptidyl-tRNA. Our findings revealed a so far unknown functional role of a tdR thus further enlarging the functional heterogeneity of this emerging class of ribo-regulators.

A Novel Caenorhabditis Elegans Proteinopathy Model Shows Changes in mRNA Translational Frameshifting During Aging.

BACKGROUND/AIMS: Regulation of mRNA translation is central to protein homeostasis and is optimized for speed and accuracy. Spontaneous recoding events occur virtually at any codon but at very low frequency and are commonly assumed to increase as the cell ages. METHODS: Here, we leveraged the polyglutamine(polyQ)-frameshifting model of huntingtin exon 1 with CAG repeat length in the pathological range (Htt51Q), which undergoes enhanced non-programmed translational -1 frameshifting. RESULTS: In body muscle cells of Caenorhabditis elegans, -1 frameshifting occured at the onset of expression of the zero-frame product, correlated with mRNA level of the non-frameshifted expression and formed aggregates correlated with reduced motility in C. elegans. Spontaneous frameshifting was modulated by IFG-1, the homologue of the nutrient-responsive eukaryotic initiation factor 4G (eIF4G), under normal growth conditions and NSUN-5, a conserved ribosomal RNA methyltransferase, under osmotic stress. CONCLUSION: Our results suggest that frameshifting and aggregation occur at even early stages of development and, because of their intrinsic stability, may persist and accelerate the onset of age-related proteinopathies.

Age-Induced Changes in White, Brite, and Brown Adipose Depots: A Mini-Review.

Aging is a time-related process of functional decline at organelle, cellular, tissue, and organismal level that ultimately limits life. Cellular senescence is a state of permanent growth arrest in response to stress and one of the major drivers of aging and age-related disorders. Senescent cells accumulate with age, and removal of these cells delays age-related disorders in different tissues and prolongs healthy lifespan. One of the most studied aging mechanisms is the accumulation of reactive oxygen species damage in cells, organs, and organisms over time. Elevated oxidative stress is also found in metabolic diseases such as obesity, metabolic syndrome and associated disorders. Moreover, dysregulation of the energy homeostasis is also associated with aging, and many age-related genes also control energy metabolism, with the adipose organ, comprising white, brite, and brown adipocytes, as an important metabolic player in the regulation of whole-body energy homeostasis. This review summarizes transformations in the adipose organ upon aging and cellular senescence and sheds light on the reallocation of fat mass between adipose depots, on the metabolism of white and brown adipose tissue, on the regenerative potential and adipogenic differentiation capacity of preadipocytes, and on alterations in mitochondria and bioenergetics. In conclusion, the aging process is a lifelong, creeping process with gradual decline in (pre-)adipocyte function over time. Thus, slowing down the accumulation of (pre-)adipocyte damage and dysfunction, removal of senescent preadipocytes as well as blocking deleterious compounds of the senescent secretome are protective measures to maintain a lasting state of health at old age.

Elimination of damaged mitochondria during UVB-induced senescence is orchestrated by NIX-dependent mitophagy.

Skin aging is the result of two types of aging, "intrinsic aging" an inevitable consequence of physiologic and genetically determined changes and "extrinsic aging," which is dependent on external factors such as exposure to sunlight, smoking, and dietary habits. UVB causes skin injury through the generation of free radicals and other oxidative byproducts, also contributing to DNA damage. Appearance and accumulation of senescent cells in the skin are considered one of the hallmarks of aging in this tissue. Mitochondria play an important role for the development of cellular senescence, in particular stress-induced senescence of human cells. However, many aspects of mitochondrial physiology relevant to cellular senescence and extrinsic skin aging remain to be unraveled. Here, we demonstrate that mitochondria damaged by UVB irradiation of human dermal fibroblasts (HDF) are eliminated by NIX-dependent mitophagy and that this process is important for cell survival under these conditions. Additionally, UVB-irradiation of human dermal fibroblasts (HDF) induces the shedding of extracellular vesicles (EVs), and this process is significantly enhanced in UVB-irradiated NIX-depleted cells. Our findings establish NIX as the main mitophagy receptor in the process of UVB-induced senescence and suggest the release of EVs as an alternative mechanism of mitochondrial quality control in HDF.

Senopathies-Diseases Associated with Cellular Senescence.

Cellular senescence describes a stable cell cycle arrest state with a characteristic phenotype. Senescent cells accumulate in the human body during normal aging, limiting the lifespan and promoting aging-related, but also several non-related, pathologies. We propose to refer to all diseases whose pathogenesis or progression is associated with cellular senescence as "senopathies". Targeting senescent cells with senolytics or senomorphics is likely to mitigate these pathologies. Examples of senopathies include cardiovascular, metabolic, musculoskeletal, liver, kidney, and lung diseases and neurodegeneration. For all these pathologies, animal studies provide clear mechanistic evidence for a connection between senescent cell accumulation and disease progression. The major persisting challenge in developing novel senotherapies is the heterogeneity of senescence phenotypes, causing a lack of universal biomarkers and difficulties in discriminating senescent from non-senescent cells.

The p-rpS6-zone delineates wounding responses and the healing process.

The spatial boundaries of tissue response to wounding are unknown. Here, we show that in mammals, the ribosomal protein S6 (rpS6) is phosphorylated in response to skin injury, forming a zone of activation surrounding the region of the initial insult. This p-rpS6-zone forms within minutes after wounding and is present until healing is complete. The zone is a robust marker of healing as it encapsulates features of the healing process, including proliferation, growth, cellular senescence, and angiogenesis. A mouse model that is unable to phosphorylate rpS6 shows an initial acceleration of wound closure, but results in impaired healing, identifying p-rpS6 as a modulator but not a driver of healing. Finally, the p-rpS6-zone accurately reports on the status of dermal vasculature and the effectiveness of healing, visually dividing an otherwise homogeneous tissue into regions with distinct properties.

The epitranscriptome in ageing and stress resistance: A systematic review.

Modifications of RNA, collectively called the "epitranscriptome", might provide novel biomarkers and innovative targets for interventions in geroscience but are just beginning to be studied in the context of ageing and stress resistance. RNA modifications modulate gene expression by affecting translation initiation and speed, miRNA binding, RNA stability, and RNA degradation. Nonetheless, the precise underlying molecular mechanisms and physiological consequences of most alterations of the epitranscriptome are still only poorly understood. We here systematically review different types of modifications of rRNA, tRNA and mRNA, the methodology to analyze them, current challenges in the field, and human disease associations. Furthermore, we compiled evidence for a connection between individual enzymes, which install RNA modifications, and lifespan in yeast, worm and fly. We also included resistance to different stressors and competitive fitness as search criteria for genes potentially relevant to ageing. Promising candidates identified by this approach include RCM1/NSUN5, RRP8, and F33A8.4/ZCCHC4 that introduce base methylations in rRNA, the methyltransferases DNMT2 and TRM9/ALKBH8, as well as factors involved in the thiolation or A to I editing in tRNA, and finally the m6A machinery for mRNA.

The role of lipid-based signalling in wound healing and senescence.

Lipid-based signalling modulates several cellular processes and intercellular communication during wound healing and tissue regeneration. Bioactive lipids include but are not limited to the diverse group of eicosanoids, phospholipids, and extracellular vesicles and mediate the attraction of immune cells, initiation of inflammatory responses, and their resolution. In aged individuals, wound healing and tissue regeneration are greatly impaired, resulting in a delayed healing process and non-healing wounds. Senescent cells accumulate with age in vivo, preferably at sites implicated in age-associated pathologies and their elimination was shown to alleviate many age-associated diseases and disorders. In contrast to these findings, the transient presence of senescent cells in the process of wound healing exerts beneficial effects and limits fibrosis. Hence, clearance of senescent cells during wound healing was repeatedly shown to delay wound closure in vivo. Recent findings established a dysregulated synthesis of eicosanoids, phospholipids and extracellular vesicles as part of the senescent phenotype. This intriguing connection between cellular senescence, lipid-based signalling, and the process of wound healing and tissue regeneration prompts us to compile the current knowledge in this review and propose future directions for investigation.

Promises and challenges of senolytics in skin regeneration, pathology and ageing.

The research of the last two decades has defined a crucial role of cellular senescence in both the physiology and pathology of skin, and senescent cells have been detected in conditions including development, regeneration, aging, and disease. The pathophysiology of cellular senescence in skin is complex as the phenotype of senescence pertains to several different cell types including fibroblasts, keratinocytes and melanocytes, among others. Paradoxically, the transient presence of senescent cells is believed to be beneficial in the context of development and wound healing, while the chronic presence of senescent cells is detrimental in the context of aging, diseases, and chronic wounds, which afflict predominantly the elderly. Identifying strategies to prevent senescence induction or reduce senescent burden in the skin could broadly benefit the aging population. Senolytics, drugs known to specifically eliminate senescent cells while preserving non-senescent cells, are being intensively studied for use in the clinical setting. Here, we review recent research on skin senescence, on the methods for the detection of senescent cells and describe promises and challenges related to the application of senolytic drugs. This article is part of the Special Issue - Senolytics - Edited by Joao Passos and Diana Jurk.

Size changes in miR­21 knockout mice: Geometric morphometrics on teeth, alveolar bone and mandible.

MicroRNA­21 (miR­21) is a small non­coding RNA that is differentially expressed during tooth development, particularly during amelogenesis. Although orthodontic tooth movement and the innate immune response are impaired, miR­21 knockout mice demonstrate no obvious skeletal phenotype. However, the consequence of miR­21 knockout on tooth phenotype and corresponding alveolar bone is unknown. The current study utilized landmark­based geometric morphometrics to identify anatomical dissimilarities of the three lower and upper molars, and the corresponding alveolar bone, in miR­21 knockout and wild­type control mice. The anatomical structures were visualized by microcomputer tomography. A total of 36 and 38 landmarks were placed on mandibular and maxillary molars, respectively. For the alveolar bone, 16 landmarks were selected on both anatomical sites. General Procrustes analysis revealed significantly smaller molars and dimensions of the alveolar bone in the mandible of the miR­21 knockout mice when compared with wild­type controls (P=0.03 and P=0.04, respectively). The overall dimension of the mandible was reduced by the lack of miR­21 (P=0.02). In the maxilla, the dimension of the alveolar bone was significant (P=0.02); however, this was not observed in the molars (P=0.36). Based on principal component analysis, no changes in shape for any of the anatomical sites were observed. Dental and skeletal jaw length were calculated and no prognathism was identified. However, the fluctuating asymmetry of the molars in the mandible and the maxilla was reduced in the miR­21 knockout mice by 38 and 27%, respectively. Taken together, the results of the present study revealed that the molars in the mandible and the dimension of the respective alveolar bone were smaller in miR­21 mice compared with wild­type littermates, suggesting that miR­21 influences tooth development.

Targeting cellular senescence based on interorganelle communication, multilevel proteostasis, and metabolic control.

Cellular senescence, a stable cell division arrest caused by severe damage and stress, is a hallmark of aging in vertebrates including humans. With progressing age, senescent cells accumulate in a variety of mammalian tissues, where they contribute to tissue aging, identifying cellular senescence as a major target to delay or prevent aging. There is an increasing demand for the discovery of new classes of small molecules that would either avoid or postpone cellular senescence by selectively eliminating senescent cells from the body (i.e., 'senolytics') or inactivating/switching damage-inducing properties of senescent cells (i.e., 'senostatics/senomorphics'), such as the senescence-associated secretory phenotype. Whereas compounds with senolytic or senostatic activity have already been described, their efficacy and specificity has not been fully established for clinical use yet. Here, we review mechanisms of senescence that are related to mitochondria and their interorganelle communication, and the involvement of proteostasis networks and metabolic control in the senescent phenotype. These cellular functions are associated with cellular senescence in in vitro and in vivo models but have not been fully exploited for the search of new compounds to counteract senescence yet. Therefore, we explore possibilities to target these mechanisms as new opportunities to selectively eliminate and/or disable senescent cells with the aim of tissue rejuvenation. We assume that this research will provide new compounds from the chemical space which act as mimetics of caloric restriction, modulators of calcium signaling and mitochondrial physiology, or as proteostasis optimizers, bearing the potential to counteract cellular senescence, thereby allowing healthy aging.

Heparin-binding motif mutations of human diamine oxidase allow the development of a first-in-class histamine-degrading biopharmaceutical.

Background: Excessive plasma histamine concentrations cause symptoms in mast cell activation syndrome, mastocytosis, or anaphylaxis. Anti-histamines are often insufficiently efficacious. Human diamine oxidase (hDAO) can rapidly degrade histamine and therefore represents a promising new treatment strategy for conditions with pathological histamine concentrations. Methods: Positively charged amino acids of the heparin-binding motif of hDAO were replaced with polar serine or threonine residues. Binding to heparin and heparan sulfate, cellular internalization and clearance in rodents were examined. Results: Recombinant hDAO is rapidly cleared from the circulation in rats and mice. After mutation of the heparin-binding motif, binding to heparin and heparan sulfate was strongly reduced. The double mutant rhDAO-R568S/R571T showed minimal cellular uptake. The short α-distribution half-life of the wildtype protein was eliminated, and the clearance was significantly reduced in rodents. Conclusions: The successful decrease in plasma clearance of rhDAO by mutations of the heparin-binding motif with unchanged histamine-degrading activity represents the first step towards the development of rhDAO as a first-in-class biopharmaceutical to effectively treat diseases characterized by excessive histamine concentrations in plasma and tissues. Funding: Austrian Science Fund (FWF) Hertha Firnberg program grant T1135 (EG); Sigrid Juselius Foundation, Medicinska Understödsförening Liv och Hälsa rft (TAS and SeV).

Epilipidomics of Senescent Dermal Fibroblasts Identify Lysophosphatidylcholines as Pleiotropic Senescence-Associated Secretory Phenotype (SASP) Factors.

During aging, skin accumulates senescent cells. The transient presence of senescent cells, followed by their clearance by the immune system, is important in tissue repair and homeostasis. The persistence of senescent cells that evade clearance contributes to the age-related deterioration of the skin. The senescence-associated secretory phenotype of these cells contains immunomodulatory molecules that facilitate clearance but also promote chronic damage. Here, we investigated the epilipidome-the oxidative modifications of phospholipids-of senescent dermal fibroblasts, because these molecules are among the bioactive lipids that were recently identified as senescence-associated secretory phenotype factors. Using replicative- and stress- induced senescence protocols, we identified lysophosphatidylcholines as universally elevated in senescent fibroblasts, whereas other oxidized lipids displayed a pattern that was characteristic for the used senescence protocol. When we tested the lysophosphatidylcholines for senescence-associated secretory phenotype activity, we found that they elicit chemokine release in nonsenescent fibroblasts but also interfere with toll-like receptor 2 and 6/CD36 signaling and phagocytic capacity in macrophages. Using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry imaging, we localized two lysophosphatidylcholine species in aged skin. This suggests that lysophospholipids may facilitate immune evasion and low-grade chronic inflammation in skin aging.

Raman fingerprints as promising markers of cellular senescence and aging.

Due to our aging population, understanding of the underlying molecular mechanisms constantly gains more and more importance. Senescent cells, defined by being irreversibly growth arrested and associated with a specific gene expression and secretory pattern, accumulate with age and thus contribute to several age-related diseases. However, their specific detection, especially in vivo, is still a major challenge. Raman microspectroscopy is able to record biochemical fingerprints of cells and tissues, allowing a distinction between different cellular states, or between healthy and cancer tissue. Similarly, Raman microspectroscopy was already successfully used to distinguish senescent from non-senescent cells, as well as to investigate other molecular changes that occur at cell and tissue level during aging. This review is intended to give an overview about various applications of Raman microspectroscopy to study aging, especially in the context of detecting senescent cells.

The Skin Epilipidome in Stress, Aging, and Inflammation.

Lipids are highly diverse biomolecules crucial for the formation and function of cellular membranes, for metabolism, and for cellular signaling. In the mammalian skin, lipids additionally serve for the formation of the epidermal barrier and as surface lipids, together regulating permeability, physical properties, acidification and the antimicrobial defense. Recent advances in accuracy and specificity of mass spectrometry have allowed studying enzymatic and non-enzymatic modifications of lipids-the epilipidome-multiplying the known diversity of molecules in this class. As the skin is an organ that is frequently exposed to oxidative-, chemical- and thermal stress, and to injury and inflammation, it is an ideal organ to study epilipidome dynamics, their causes, and their biological consequences. Recent studies uncover loss or gain in biological function resulting from either specific modifications or the sum of the modifications of lipids. These studies suggest an important role for the epilipidome in stress responses and immune regulation in the skin. In this minireview we provide a short survey of the recent developments on causes and consequences of epilipidomic changes in the skin or in cell types that reside in the skin.

The ribosomal RNA m5C methyltransferase NSUN-1 modulates healthspan and oogenesis in Caenorhabditis elegans.

Our knowledge about the repertoire of ribosomal RNA modifications and the enzymes responsible for installing them is constantly expanding. Previously, we reported that NSUN-5 is responsible for depositing m5C at position C2381 on the 26S rRNA in Caenorhabditis elegans. Here, we show that NSUN-1 is writing the second known 26S rRNA m5C at position C2982. Depletion of nsun-1 or nsun-5 improved thermotolerance and slightly increased locomotion at midlife, however, only soma-specific knockdown of nsun-1 extended lifespan. Moreover, soma-specific knockdown of nsun-1 reduced body size and impaired fecundity, suggesting non-cell-autonomous effects. While ribosome biogenesis and global protein synthesis were unaffected by nsun-1 depletion, translation of specific mRNAs was remodeled leading to reduced production of collagens, loss of structural integrity of the cuticle, and impaired barrier function. We conclude that loss of a single enzyme required for rRNA methylation has profound and highly specific effects on organismal development and physiology.

miRNA-21 deficiency impairs alveolar socket healing in mice.

BACKGROUND: MicroRNAs (miRNAs) are small noncoding RNAs demonstrated as critical post-transcriptional modulators in dental tissues and bone regeneration, particularly miR-21-5p. However, the role of miR-21-5p in the healing of alveolar sockets following tooth extraction remains unknown. In this study we evaluated the influence of miR-21-5p in the healing of alveolar socket after tooth extraction. METHODS: Eight miR-21-5p knockout mice and eight littermate controls underwent tooth extraction of the upper right incisor. After a healing period of 14 days microCT and histological analyses were performed. RESULTS: MicroCT analysis showed that the percentage of bone in the extraction socket was significantly higher in the control group than in the miR-21 knockout mice; either in the coronal (39.0%, CI 31.8 to 48.0 versus 23.0%, CI 17.8 to 35.2, P = 0.03) or in the middle part of the alveolar socket (56.0%, CI 50.9 to 62.5 versus 43.5% CI 28.6 to 54.6, P = 0.03). These differences were not noted in the apical part of the extraction socket. Histological analysis supported the microCT findings. Newly bone volume per tissue volume (BV/TV) was significantly higher in the control group when compared to miR-21 knockout mice, 27.4% (CI 20.6 to 32.9) versus 19.0% (CI 14.7 to 21.5, P < 0.05), respectively. Surprisingly, no evident signs of buccal bone resorption were observed in both groups. CONCLUSION: Despite the limitation of one observation period, these findings suggest that miR-21-5p delays the early healing of alveolar socket following tooth extraction. Whether miR-21-5p is essential for healing of alveolar sockets remains to be elucidated.