Hallmarks of aging: An expanding universe.

Aging is driven by hallmarks fulfilling the following three premises: (1) their age-associated manifestation, (2) the acceleration of aging by experimentally accentuating them, and (3) the opportunity to decelerate, stop, or reverse aging by therapeutic interventions on them. We propose the following twelve hallmarks of aging: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, disabled macroautophagy, deregulated nutrient-sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis. These hallmarks are interconnected among each other, as well as to the recently proposed hallmarks of health, which include organizational features of spatial compartmentalization, maintenance of homeostasis, and adequate responses to stress.

Hallmarks of Health.

Health is usually defined as the absence of pathology. Here, we endeavor to define health as a compendium of organizational and dynamic features that maintain physiology. The biological causes or hallmarks of health include features of spatial compartmentalization (integrity of barriers and containment of local perturbations), maintenance of homeostasis over time (recycling and turnover, integration of circuitries, and rhythmic oscillations), and an array of adequate responses to stress (homeostatic resilience, hormetic regulation, and repair and regeneration). Disruption of any of these interlocked features is broadly pathogenic, causing an acute or progressive derailment of the system coupled to the loss of numerous stigmata of health.

Carbotoxicity-Noxious Effects of Carbohydrates.

Modern nutrition is often characterized by the excessive intake of different types of carbohydrates ranging from digestible polysaccharides to refined sugars that collectively mediate noxious effects on human health, a phenomenon that we refer to as "carbotoxicity." Epidemiological and experimental evidence combined with clinical intervention trials underscore the negative impact of excessive carbohydrate uptake, as well as the beneficial effects of reducing carbs in the diet. We discuss the molecular, cellular, and neuroendocrine mechanisms that link exaggerated carbohydrate intake to disease and accelerated aging as we outline dietary and pharmacologic strategies to combat carbotoxicity.

Metabolic Control of Longevity.

Several metabolic alterations accumulate over time along with a reduction in biological fitness, suggesting the existence of a "metabolic clock" that controls aging. Multiple inborn defects in metabolic circuitries accelerate aging, whereas genetic loci linked to exceptional longevity influence metabolism. Each of the nine hallmarks of aging is connected to undesirable metabolic alterations. The main features of the "westernized" lifestyle, including hypercaloric nutrition and sedentariness, can accelerate aging as they have detrimental metabolic consequences. Conversely, lifespan-extending maneuvers including caloric restriction impose beneficial pleiotropic effects on metabolism. The introduction of strategies that promote metabolic fitness may extend healthspan in humans.

Progeria: a paradigm for translational medicine.

Rare diseases are powerful windows into biological processes and can serve as models for the development of therapeutic strategies. The progress made on the premature aging disorder Progeria is a shining example of the impact that studies of rare diseases can have.

The hallmarks of aging.

Aging is characterized by a progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death. This deterioration is the primary risk factor for major human pathologies, including cancer, diabetes, cardiovascular disorders, and neurodegenerative diseases. Aging research has experienced an unprecedented advance over recent years, particularly with the discovery that the rate of aging is controlled, at least to some extent, by genetic pathways and biochemical processes conserved in evolution. This Review enumerates nine tentative hallmarks that represent common denominators of aging in different organisms, with special emphasis on mammalian aging. These hallmarks are: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication. A major challenge is to dissect the interconnectedness between the candidate hallmarks and their relative contributions to aging, with the final goal of identifying pharmaceutical targets to improve human health during aging, with minimal side effects.

Immunosuppression by Mutated Calreticulin Released from Malignant Cells.

Mutations affecting exon 9 of the CALR gene lead to the generation of a C-terminally modified calreticulin (CALR) protein that lacks the KDEL endoplasmic reticulum (ER) retention signal and consequently mislocalizes outside of the ER where it activates the thrombopoietin receptor in a cell-autonomous fashion, thus driving myeloproliferative diseases. Here, we used the retention using selective hooks (RUSH) assay to monitor the trafficking of CALR. We found that exon-9-mutated CALR was released from cells in response to the biotin-mediated detachment from its ER-localized hook, in vitro and in vivo. Cellular CALR release was confirmed in suitable mouse models bearing exon-9-mutated hematopoietic systems or tumors. Extracellular CALR mediated immunomodulatory effects and inhibited the phagocytosis of dying cancer cells by dendritic cells (DC), thereby suppressing antineoplastic immune responses elicited by chemotherapeutic agents or by PD-1 blockade. Altogether, our results demonstrate paracrine immunosuppressive effects for exon-9-mutated CALR.

New roles for mitochondrial proteases in health, ageing and disease.

Recent advances in mitochondrial biology have revealed the high diversity and complexity of proteolytic enzymes that regulate mitochondrial function. We have classified mitochondrial proteases, or mitoproteases, on the basis of their function and location, and defined the human mitochondrial degradome as the complete set of mitoproteases that are encoded by the human genome. In addition to their nonspecific degradative functions, mitoproteases perform highly regulated proteolytic reactions that are important in mitochondrial function, integrity and homeostasis. These include protein synthesis, quality control, mitochondrial biogenesis and dynamics, mitophagy and apoptosis. Impaired or dysregulated function of mitoproteases is associated with ageing and with many pathological conditions such as neurodegenerative disorders, metabolic syndromes and cancer. A better understanding of the mitochondrial proteolytic landscape and its modulation may contribute to improving human lifespan and 'healthspan'.

Autophagy-linked plasma and lysosomal membrane protein PLAC8 is a key host factor for SARS-CoV-2 entry into human cells.

Better understanding on interactions between SARS-CoV-2 and host cells should help to identify host factors that may be targetable to combat infection and COVID-19 pathology. To this end, we have conducted a genome-wide CRISPR/Cas9-based loss-of-function screen in human lung cancer cells infected with SARS-CoV-2-pseudotyped lentiviruses. Our results recapitulate many findings from previous screens that used full SARS-CoV-2 viruses, but also unveil two novel critical host factors: the lysosomal efflux transporter SPNS1 and the plasma and lysosomal membrane protein PLAC8. Functional experiments with full SARS-CoV-2 viruses confirm that loss-of-function of these genes impairs viral entry. We find that PLAC8 is a key limiting host factor, whose overexpression boosts viral infection in eight different human lung cancer cell lines. Using single-cell RNA-Seq data analyses, we demonstrate that PLAC8 is highly expressed in ciliated and secretory cells of the respiratory tract, as well as in gut enterocytes, cell types that are highly susceptible to SARS-CoV-2 infection. Proteomics and cell biology studies suggest that PLAC8 and SPNS1 regulate the autophagolysosomal compartment and affect the intracellular fate of endocytosed virions.

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.

Endothelial-to-Mesenchymal Transition Contributes to Accelerated Atherosclerosis in Hutchinson-Gilford Progeria Syndrome.

BACKGROUND: Atherosclerosis is the main medical problem in Hutchinson-Gilford progeria syndrome, a rare premature aging disorder caused by the mutant lamin-A protein progerin. Recently, we found that limiting progerin expression to vascular smooth muscle cells (VSMCs) is sufficient to hasten atherosclerosis and death in Apoe-deficient mice. However, the impact of progerin-driven VSMC defects on endothelial cells (ECs) remained unclear. METHODS: Apoe- or Ldlr-deficient C57BL/6J mice with ubiquitous, VSMC-, EC- or myeloid-specific progerin expression fed a normal or high-fat diet were used to study endothelial phenotype during Hutchinson-Gilford progeria syndrome-associated atherosclerosis. Endothelial permeability to low-density lipoproteins was assessed by intravenous injection of fluorescently labeled human low-density lipoprotein and confocal microscopy analysis of the aorta. Leukocyte recruitment to the aortic wall was evaluated by en face immunofluorescence. Endothelial-to-mesenchymal transition (EndMT) was assessed by quantitative polymerase chain reaction and RNA sequencing in the aortic intima and by immunofluorescence in aortic root sections. TGFß (transforming growth factor ß) signaling was analyzed by multiplex immunoassay in serum, by Western blot in the aorta, and by immunofluorescence in aortic root sections. The therapeutic benefit of TGFß1/SMAD3 pathway inhibition was evaluated in mice by intraperitoneal injection of SIS3 (specific inhibitor of SMAD3), and vascular phenotype was assessed by Oil Red O staining, histology, and immunofluorescence in the aorta and the aortic root. RESULTS: Both ubiquitous and VSMC-specific progerin expression in Apoe-null mice provoked alterations in aortic ECs, including increased permeability to low-density lipoprotein and leukocyte recruitment. Atherosclerotic lesions in these progeroid mouse models, but not in EC- and myeloid-specific progeria models, contained abundant cells combining endothelial and mesenchymal features, indicating extensive EndMT triggered by dysfunctional VSMCs. Accordingly, the intima of ubiquitous and VSMC-specific progeroid models at the onset of atherosclerosis presented increased expression of EndMT-linked genes, especially those specific to fibroblasts and extracellular matrix. Aorta in both models showed activation of the TGFß1/SMAD3 pathway, a major trigger of EndMT, and treatment of VSMC-specific progeroid mice with SIS3 alleviated the aortic phenotype. CONCLUSIONS: Progerin-induced VSMC alterations promote EC dysfunction and EndMT through TGFß1/SMAD3, identifying this process as a candidate target for Hutchinson-Gilford progeria syndrome treatment. These findings also provide insight into the complex role of EndMT during atherogenesis.

Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

The U1 spliceosomal RNA is recurrently mutated in multiple cancers.

Cancers are caused by genomic alterations known as drivers. Hundreds of drivers in coding genes are known but, to date, only a handful of noncoding drivers have been discovered-despite intensive searching1,2. Attention has recently shifted to the role of altered RNA splicing in cancer; driver mutations that lead to transcriptome-wide aberrant splicing have been identified in multiple types of cancer, although these mutations have only been found in protein-coding splicing factors such as splicing factor 3b subunit 1 (SF3B1)3-6. By contrast, cancer-related alterations in the noncoding component of the spliceosome-a series of small nuclear RNAs (snRNAs)-have barely been studied, owing to the combined challenges of characterizing noncoding cancer drivers and the repetitive nature of snRNA genes1,7,8. Here we report a highly recurrent A>C somatic mutation at the third base of U1 snRNA in several types of tumour. The primary function of U1 snRNA is to recognize the 5' splice site via base-pairing. This mutation changes the preferential A-U base-pairing between U1 snRNA and the 5' splice site to C-G base-pairing, and thus creates novel splice junctions and alters the splicing pattern of multiple genes-including known drivers of cancer. Clinically, the A>C mutation is associated with heavy alcohol use in patients with hepatocellular carcinoma, and with the aggressive subtype of chronic lymphocytic leukaemia with unmutated immunoglobulin heavy-chain variable regions. The mutation in U1 snRNA also independently confers an adverse prognosis to patients with chronic lymphocytic leukaemia. Our study demonstrates a noncoding driver in spliceosomal RNAs, reveals a mechanism of aberrant splicing in cancer and may represent a new target for treatment. Our findings also suggest that driver discovery should be extended to a wider range of genomic regions.

Comparative genomics of mortal and immortal cnidarians unveils novel keys behind rejuvenation.

Turritopsis dohrnii is the only metazoan able to rejuvenate repeatedly after its medusae reproduce, hinting at biological immortality and challenging our understanding of aging. We present and compare whole-genome assemblies of T. dohrnii and the nonimmortal Turritopsis rubra using automatic and manual annotations, together with the transcriptome of life cycle reversal (LCR) process of T. dohrnii. We have identified variants and expansions of genes associated with replication, DNA repair, telomere maintenance, redox environment, stem cell population, and intercellular communication. Moreover, we have found silencing of polycomb repressive complex 2 targets and activation of pluripotency targets during LCR, which points to these transcription factors as pluripotency inducers in T. dohrnii. Accordingly, we propose these factors as key elements in the ability of T. dohrnii to undergo rejuvenation.

Aging and cancer.

Aging and cancer exhibit apparent links that we will examine in this review. The null hypothesis that aging and cancer coincide because both are driven by time, irrespective of the precise causes, can be confronted with the idea that aging and cancer share common mechanistic grounds that are referred to as 'hallmarks'. Indeed, several hallmarks of aging also contribute to carcinogenesis and tumor progression, but some of the molecular and cellular characteristics of aging may also reduce the probability of developing lethal cancer, perhaps explaining why very old age (> 90 years) is accompanied by a reduced incidence of neoplastic diseases. We will also discuss the possibility that the aging process itself causes cancer, meaning that the time-dependent degradation of cellular and supracellular functions that accompanies aging produces cancer as a byproduct or 'age-associated disease'. Conversely, cancer and its treatment may erode health and drive the aging process, as this has dramatically been documented for cancer survivors diagnosed during childhood, adolescence, and young adulthood. We conclude that aging and cancer are connected by common superior causes including endogenous and lifestyle factors, as well as by a bidirectional crosstalk, that together render old age not only a risk factor of cancer but also an important parameter that must be considered for therapeutic decisions.

Mitochondrial Safeguard: a stress response that offsets extreme fusion and protects respiratory function via flickering-induced Oma1 activation.

The connectivity of mitochondria is regulated by a balance between fusion and division. Many human diseases are associated with excessive mitochondrial connectivity due to impaired Drp1, a dynamin-related GTPase that mediates division. Here, we report a mitochondrial stress response, named mitochondrial safeguard, that adjusts the balance of fusion and division in response to increased mitochondrial connectivity. In cells lacking Drp1, mitochondria undergo hyperfusion. However, hyperfusion does not completely connect mitochondria because Opa1 and mitofusin 1, two other dynamin-related GTPases that mediate fusion, become proteolytically inactivated. Pharmacological and genetic experiments show that the activity of Oma1, a metalloprotease that cleaves Opa1, is regulated by short pulses of the membrane depolarization without affecting the overall membrane potential in Drp1-knockout cells. Re-activation of Opa1 and Mitofusin 1 in Drp1-knockout cells further connects mitochondria beyond hyperfusion, termed extreme fusion, leading to bioenergetic deficits. These findings reveal an unforeseen safeguard mechanism that prevents extreme fusion of mitochondria, thereby maintaining mitochondrial function when the balance is shifted to excessive connectivity.

TDP-43 regulates LC3ylation in neural tissue through ATG4B cryptic splicing inhibition.

Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease with a mean survival time of three years. The 97% of the cases have TDP-43 nuclear depletion and cytoplasmic aggregation in motor neurons. TDP-43 prevents non-conserved cryptic exon splicing in certain genes, maintaining transcript stability, including ATG4B, which is crucial for autophagosome maturation and Microtubule-associated proteins 1A/1B light chain 3B (LC3B) homeostasis. In ALS mice (G93A), Atg4b depletion worsens survival rates and autophagy function. For the first time, we observed an elevation of LC3ylation in the CNS of both ALS patients and atg4b-/- mouse spinal cords. Furthermore, LC3ylation modulates the distribution of ATG3 across membrane compartments. Antisense oligonucleotides (ASOs) targeting cryptic exon restore ATG4B mRNA in TARDBP knockdown cells. We further developed multi-target ASOs targeting TDP-43 binding sequences for a broader effect. Importantly, our ASO based in peptide-PMO conjugates show brain distribution post-IV administration, offering a non-invasive ASO-based treatment avenue for neurodegenerative diseases.

Correction: Regulation of ATG4B Stability by RNF5 Limits Basal Levels of Autophagy and Influences Susceptibility to Bacterial Infection.

[This corrects the article DOI: 10.1371/journal.pgen.1003007.].