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Little is known about tissue specific changes that occur with aging in humans. Using the description of 33 million histological samples we extract thousands of age- and mortality-associated features from text narratives that we call The Human Pathome (pathoage.com). Notably, we can determine when pathological aging starts at the organism and tissue level, indicating a sexual dimorphism with females aging earlier but slower and males aging later but faster. We employ unsupervised topic-modelling to identify terms and themes that predict age and mortality. As a proof of principle, we cross reference these terms in PubMed to identify nintedanib as a potential aging intervention and show that nintedanib reduces markers of cellular senescence, reduces pro-fibrotic gene pathways in senescent cells and extends the lifespan of fruit flies. Our findings pave the way for expanded exploitation of population datasets towards discovery of novel aging interventions.

SUMMARYThere is currently no established intervention for Cockayne syndrome, a disease characterized by progressive early onset neurodegeneration with features of premature aging. Here, we tested if acetyl-CoA precursors, citrate and beta-hydroxybutyrate, could reduce features of Cockayne syndrome in three model systems. We identified the gene Helicase 89B as a homologue of CSB in drosophila and found that the ketone beta-hydroxybutyrate rescued features of premature aging in Hel89B deficient flies. In mammals, loss of the citrate carrier Indy exacerbated the phenotype of Csbm/m mice which was rescued by a ketogenic diet. The rescue effect appeared to be mediated through ketone stimulated histone acetylation and facilitation of transcriptional readthrough of secondary DNA structures. These findings link a ketogenic diet with transcriptional resolution of secondary structures and DNA repair.

The risk of morbidity and mortality increases exponentially with age. Chronic inflammation, accumulation of DNA damage, dysfunctional mitochondria, and increased senescent cell load are factors contributing to this. Mechanistic investigations have revealed specific pathways and processes which, proposedly, cause age-related phenotypes such as frailty, reduced physical resilience, and multi-morbidity. Among promising treatments alleviating the consequences of aging are caloric restriction and pharmacologically targeting longevity pathways such as the mechanistic target of rapamycin (mTOR), sirtuins, and anti-apoptotic pathways in senescent cells. Regulation of these pathways and processes has revealed significant health- and lifespan extending results in animal models. Nevertheless, it remains unclear if similar results translate to humans. A requirement of translation are the development of age- and morbidity associated biomarkers as longitudinal trials are difficult and not feasible, practical, nor ethical when human life span is the endpoint. Current biomarkers and the results of anti-aging intervention studies in humans will be covered within this paper. The future of clinical trials targeting aging may be phase 2 and 3 studies with larger populations if safety and tolerability of investigated medication continues not to be a hurdle for further investigations.

DNA mismatch repair (MMR) is a highly conserved pathway that corrects both base–base mispairs and insertion-deletion loops (IDLs) generated during DNA replication. Defects in MMR have been linked to carcinogenesis and drug resistance. However, the regulation of MMR is poorly understood. Interestingly, CNOT6 is one of four deadenylase subunits in the conserved CCR4-NOT complex and it targets poly(A) tails of mRNAs for degradation. CNOT6 is overexpressed in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and androgen-independent prostate cancer cells, which suggests that an altered expression of CNOT6 may play a role in tumorigenesis. Here, we report that a depletion of CNOT6 sensitizes human U2OS cells to N-methyl-N′nitro-N-nitrosoguanidine (MNNG) and leads to enhanced apoptosis. We also demonstrate that the depletion of CNOT6 upregulates MMR and decreases the mutation frequency in MMR-proficient cells. Furthermore, the depletion of CNOT6 increases the stability of mRNA transcripts from MMR genes, leading to the increased expression of MMR proteins. Our work provides insight into a novel CNOT6-dependent mechanism for regulating MMR.

Aging is the single largest risk factor for most chronic diseases, and thus possesses large socioeconomic interest to continuously aging societies. Consequently, the field of aging research is expanding alongside a growing focus from the industry and investors in aging research. This year's 8th Annual Aging Research and Drug Discovery (ARDD) meeting was organized as a hybrid meeting from August 30th to September 3rd 2021 with more than 130 attendees participating on-site at the Ceremonial Hall at University of Copenhagen, Denmark, and 1800 engaging online. The conference comprised of presentations from 75 speakers focusing on new research in topics including mechanisms of aging and how these can be modulated as well as the use of AI and new standards of practices within aging research. This year, a longevity workshop was included to build stronger connections with the clinical community., Aging, 14 (2), ISSN:1945-4589

Cellular senescence is an important factor in aging and many age-related diseases, but understanding its role in health is challenging due to the lack of exclusive or universal markers. Using neural networks, we predict senescence from the nuclear morphology of human fibroblasts with up to 95% accuracy, and investigate murine astrocytes, murine neurons, and fibroblasts with premature aging in culture. After generalizing our approach, the predictor recognizes higher rates of senescence in p21-positive and ethynyl-2’-deoxyuridine (EdU)-negative nuclei in tissues and shows an increasing rate of senescent cells with age in H&E-stained murine liver tissue and human dermal biopsies. Evaluating medical records reveals that higher rates of senescent cells correspond to decreased rates of malignant neoplasms and increased rates of osteoporosis, osteoarthritis, hypertension and cerebral infarction. In sum, we show that morphological alterations of the nucleus can serve as a deep learning predictor of senescence that is applicable across tissues and species and is associated with health outcomes in humans.

Cellular senescence is a critical component of aging and many age-related diseases, but understanding its role in human health is challenging in part due to the lack of exclusive or universal markers. Using neural networks, we achieve high accuracy in predicting senescence state and type from the nuclear morphology of DAPI-stained human fibroblasts, murine astrocytes, murine neurons, and fibroblasts derived from premature aging diseases in culture. After generalizing this approach, the predictor recognizes an increasing rate of senescent cells with age in H&E-stained murine liver tissue and human dermal biopsies, suggesting that alterations in nuclear morphology is a universal feature of senescence. Evaluating corresponding medical records reveals that individuals with a higher rate of senescent cells have a significantly decreased rate of malignant neoplasms, lending support for the protective role of senescence in limiting cancer development. Additionally, we find a positive association with lower significance for other conditions, including osteoporosis, osteoarthritis, hypertension, cerebral infarction, hyperlipidemia, and hypercholesteremia. In sum, we introduce a predictor of cellular senescence based on nuclear morphology that is applicable across tissues and species and is associated with health outcomes in humans.

An increasing aging population poses a significant challenge to societies worldwide. A better understanding of the molecular, cellular, organ, tissue, physiological, psychological, and even sociological changes that occur with aging is needed in order to treat age-associated diseases. The field of aging research is rapidly expanding with multiple advances transpiring in many previously disconnected areas. Several major pharmaceutical, biotechnology, and consumer companies made aging research a priority and are building internal expertise, integrating aging research into traditional business models and exploring new go-to-market strategies. Many of these efforts are spearheaded by the latest advances in artificial intelligence, namely deep learning, including generative and reinforcement learning. To facilitate these trends, the Center for Healthy Aging at the University of Copenhagen and Insilico Medicine are building a community of Key Opinion Leaders (KOLs) in these areas and launched the annual conference series titled "Aging Research and Drug Discovery (ARDD)" held in the capital of the pharmaceutical industry, Basel, Switzerland (www.agingpharma.org). This ARDD collection contains summaries from the 6th annual meeting that explored aging mechanisms and new interventions in age-associated diseases. The 7th annual ARDD exhibition will transpire 2nd-4th of September, 2020, in Basel.

Maintenance of genomic integrity involves cellular processes tailored to specific types of DNA damage. Monogenic disorders in DNA-repair pathways lead to a spectrum of clinical phenotypes that are not always correlated with our understanding of the affected repair pathway.

Maintaining mitochondrial health is emerging as a keystone in aging and associated diseases. The selective degradation of mitochondria by mitophagy is of particular importance in keeping a pristine mitochondrial pool. Indeed, inherited monogenic diseases with defects in mitophagy display complex multisystem pathologies but particularly progressive neurodegeneration. Fortunately, therapies are being developed that target mitophagy allowing new hope for treatments for previously incurable diseases. Herein, we describe mitophagy and associated diseases, coin the term mitophaging and describe new small molecule interventions that target different steps in the mitophagic pathway. Consequently, several age-associated diseases may be treated by targeting mitophagy.

Ageing is arguably the most complex phenotype that occurs in humans. To understand and treat ageing as well as associated diseases, highly specialised technologies are emerging that reveal critical insight into the underlying mechanisms and provide new hope for previously untreated diseases. Herein, we describe the latest developments in cutting edge technologies applied across the field of ageing research. We cover emerging model organisms, high-throughput methodologies and machine-driven approaches. In all, this review will give you a glimpse of what will be pushing the field onwards and upwards.

Aging is emerging as a druggable target with growing interest from academia, industry and investors. New technologies such as artificial intelligence and advanced screening techniques, as well as a strong influence from the industry sector may lead to novel discoveries to treat age-related diseases. The present review summarizes presentations from the 7th Annual Aging Research and Drug Discovery (ARDD) meeting, held online on the 1st to 4th of September 2020. The meeting covered topics related to new methodologies to study aging, knowledge about basic mechanisms of longevity, latest interventional strategies to target the aging process as well as discussions about the impact of aging research on society and economy. More than 2000 participants and 65 speakers joined the meeting and we already look forward to an even larger meeting next year. Please mark your calendars for the 8th ARDD meeting that is scheduled for the 31st of August to 3rd of September, 2021, at Columbia University, USA., Aging, 12 (24), ISSN:1945-4589

Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of CAG repeats in the ATXN3 gene leading to an elongated polyglutamine tract in the ataxin-3 protein. Previously, we demonstrated that symptoms of SCA3 are reversible in the first conditional mouse model for SCA3 directing ataxin-3 predominantly to the hindbrain. Here, we report on the effects of transgenic ataxin-3 expression in forebrain regions. Employing the Tet-off CamKII-promoter mouse line and our previously published SCA3 responder line, we generated double transgenic mice (CamKII/MJD77), which develop a neurological phenotype characterized by impairment in rotarod performance, and deficits in learning new motor tasks as well as hyperactivity. Ataxin-3 and ubiquitin-positive inclusions are detected in brains of double transgenic CamKII/MJD77 mice. After turning off the expression of pathologically expanded ataxin-3, these inclusions disappear. However, the observed phenotype could not be reversed, very likely due to pronounced apoptotic cell death in the frontal brain. Our data demonstrate that cerebellar expression is not required to induce a neurological phenotype using expanded ATXN3 as well as the pronounced sensibility of forebrain neurons for toxic ataxin-3.

Multiple interventions in the aging process have been discovered to extend the healthspan of model organisms. Both industry and academia are therefore exploring possible transformative molecules that target aging and age‐associated diseases. In this overview, we summarize the presented talks and discussion points of the 5th Annual Aging and Drug Discovery Forum 2018 in Basel, Switzerland. Here academia and industry came together, to discuss the latest progress and issues in aging research. The meeting covered talks about the mechanistic cause of aging, how longevity signatures may be highly conserved, emerging biomarkers of aging, possible interventions in the aging process and the use of artificial intelligence for aging research and drug discovery. Importantly, a consensus is emerging both in industry and academia, that molecules able to intervene in the aging process may contain the potential to transform both societies and healthcare, DB is supported by the German Research Foundation (Forschungsstipendium; BA 6276/1-1). CYE is supported by Swiss National Science Foundation [163898]. VNG is supported by grants from National Institutes of Health, and by the Russian Federation grant 14.W03.31.0012. DWL presented the results of research supported in part by research grants and funds from the National Institutes of Health, the Wisconsin Partnership Program, the Progeria Research Foundation, the American Federation for Aging Research, and the University of Wisconsin-Madison School of Medicine and Public Health and Department of Medicine, as well as the facilities and resources of the William S. Middleton Memorial Veterans Hospital. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. This work does not represent the views of the Department of Veterans Affairs or the United States Government. MSL is supported by an LUMC research fellowship and a VIDI grant from the Netherlands scientific organization (NWO- ALW-016.161.320). A.M.-M. is supported by grants from the Instituto de Salud Carlos III co-funded by Instituto de Salud Carlos III and FEdeR (CP14/00105 and PI15/00134). SM was supported by the FWO-OP/Odysseus program (42/FA010100/32/6484). SJO's current work is funded by The Glenn Award from the Glenn Foundation for Medical Research. MR is supported by the Swiss National Science Foundation and the European Union Horizon 2020 program. MSK is supported by grants from the Danish Cancer Society (#R167-A11015_001), the Independent Research Fund Denmark (#7016- 00230B) and the Novo Nordisk Foundation (NNF17OC0027812).

The article discusses new findings on the role of the 4 human WIPI proteins at the onset of macroautophagy/autophagy. New insights revealing a circuit scaffold function of WIPI β-propellers that interconnect autophagy signaling control with appropriate autophagosome formation are summarized.

Autophagy is a catabolic pathway in which the cell sequesters cytoplasmic material, including long-lived proteins, lipids and organelles, in specialized double-membrane vesicles, called autophagosomes. Subsequently, autophagosomes communicate with the lysosomal compartment and acquire acidic hydrolases for final cargo degradation. This process of partial self-eating secures the survival of eukaryotic cells during starvation periods and is critically regulated by mTORC1 (mammalian target of rapamycin complex 1). Under nutrient-poor conditions, inhibited mTORC1 permits localized PtdIns(3)P production at particular membranes that contribute to autophagosome formation. Members of the human WIPI (WD-repeat protein interacting with phosphoinositides) family fulfil an essential role as PtdIns(3)P effectors at the initiation step of autophagosome formation. In the present article, we discuss the role of human WIPIs in autophagy, and the identification of evolutionarily conserved amino acids of WIPI-1 that confer PtdIns(3)P binding downstream of mTORC1 inhibition. We also discuss the PtdIns(3)P effector function of WIPIs in the context of longevity and autophagy-related human diseases, such as cancer and neurodegeneration.

Autophagy is controlled by AMPK and mTOR, both of which associate with ULK1 and control the production of phosphatidylinositol 3-phosphate (PtdIns3P), a prerequisite for autophagosome formation. Here we report that WIPI3 and WIPI4 scaffold the signal control of autophagy upstream of PtdIns3P production and have a role in the PtdIns3P effector function of WIPI1-WIPI2 at nascent autophagosomes. In response to LKB1-mediated AMPK stimulation, WIPI4-ATG2 is released from a WIPI4-ATG2/AMPK-ULK1 complex and translocates to nascent autophagosomes, controlling their size, to which WIPI3, in complex with FIP200, also contributes. Upstream, WIPI3 associates with AMPK-activated TSC complex at lysosomes, regulating mTOR. Our WIPI interactome analysis reveals the scaffold functions of WIPI proteins interconnecting autophagy signal control and autophagosome formation. Our functional kinase screen uncovers a novel regulatory link between LKB1-mediated AMPK stimulation that produces a direct signal via WIPI4, and we show that the AMPK-related kinases NUAK2 and BRSK2 regulate autophagy through WIPI4., During autophagy, AMPK and mTOR associate with ULK1 and regulate phosphatidylinositol 3-phosphate (PtdIns3P) production that mediates autophagosome formation via WIPI proteins. Here the authors show WIPI3 and WIPI4 have a scaffolding function upstream of PtdIns3P production and have a role in the PtdIns3P effector function of WIPI1-WIPI2 at nascent autophagosomes.

Human exonuclease 1 (EXO1), a 5′→3′ exonuclease, contributes to the regulation of the cell cycle checkpoints, replication fork maintenance, and post replicative DNA repair pathways. These processes are required for the resolution of stalled or blocked DNA replication that can lead to replication stress and potential collapse of the replication fork. Failure to restart the DNA replication process can result in double-strand breaks, cell-cycle arrest, cell death, or cellular transformation. In this review, we summarize the involvement of EXO1 in the replication, DNA repair pathways, cell cycle checkpoints, and the link between EXO1 and cancer.

Autophagy is a lysosomal bulk degradation pathway for cytoplasmic cargo, such as long-lived proteins, lipids, and organelles. Induced upon nutrient starvation, autophagic degradation is accomplished by the concerted actions of autophagy-related (ATG) proteins. Here we demonstrate that two ATGs, human Atg2A and Atg14L, colocalize at cytoplasmic lipid droplets (LDs) and are functionally involved in controlling the number and size of LDs in human tumor cell lines. We show that Atg2A is targeted to cytoplasmic ADRP-positive LDs that migrate bidirectionally along microtubules. The LD localization of Atg2A was found to be independent of the autophagic status. Further, Atg2A colocalized with Atg14L under nutrient-rich conditions when autophagy was not induced. Upon nutrient starvation and dependent on phosphatidylinositol 3-phosphate [PtdIns(3)P] generation, both Atg2A and Atg14L were also specifically targeted to endoplasmic reticulum-associated early autophagosomal membranes, marked by the PtdIns(3)P effectors double-FYVE containing protein 1 (DFCP1) and WD-repeat protein interacting with phosphoinositides 1 (WIPI-1), both of which function at the onset of autophagy. These data provide evidence for additional roles of Atg2A and Atg14L in the formation of early autophagosomal membranes and also in lipid metabolism.

Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress-the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca (2+)-ATPase inhibitor thapsigargin (TG)-potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.

Eukaroytic cellular homeostasis is critically secured by autophagy, a catabolic pathway for the degradation of cytoplasmic material in the lysosomal compartment. Macroautophagy, one of the major autophagic pathway, is initiated upon PtdIns(3)P generation by activated PtdIns3KC3 in complex with Beclin 1, p150 and Atg14L. Subsequently, specific PtdIns(3)Peffector proteins permit the formation of double-membrane vesicles, autophagosomes, that sequester the cytoplasmic material. Autophagosomes then communicate and fuse with the lysosomal compartment for final cargo degradation. Members of the human WIPI family function as essential PtdIns(3)P-binding proteins during the initiation of macroautophagy downstream of PtdIns3KC3, and become membrane proteins of generated autophagosomes. Here, we discuss the function of human WIPIs and describe the WIPI puncta-formation analysis for the quantitative assessment of macroautophagy.

Summary Autophagy is a conserved degradative transport pathway. It is characterized by the formation of double-membrane autophagosomes at the phagophore assembly site (PAS). Atg18 is essential for autophagy but also for vacuole homeostasis and probably endosomal functions. This protein is basically a β-propeller, formed by seven WD40 repeats, that contains a conserved FRRG motif that binds to phosphoinositides and promotes Atg18 recruitment to the PAS, endosomes and vacuoles. However, it is unknown how Atg18 association with these organelles is regulated, as the phosphoinositides bound by this protein are present on the surface of all of them. We have investigated Atg18 recruitment to the PAS and found that Atg18 binds to Atg2 through a specific stretch of amino acids in the β-propeller on the opposite surface to the FRRG motif. As in the absence of the FRRG sequence, the inability of Atg18 to interact with Atg2 impairs its association with the PAS, causing an autophagy block. Our data provide a model whereby the Atg18 β-propeller provides organelle specificity by binding to two determinants on the target membrane.

Background: Autophagy is a cytoprotective, lysosomal degradation system regulated upon induced phosphatidylinositol 3-phosphate (PtdIns(3)P) generation by phosphatidylinositol 3-kinase class III (PtdIns3KC3) downstream of mTORC1 inhibition. The human PtdIns(3)P-binding β-propeller protein WIPI-1 accumulates at the initiation site for autophagosome formation (phagophore), functions upstream of the Atg12 and LC3 conjugation systems, and localizes at both the inner and outer membrane of generated autophagosomes. In addition, to a minor degree WIPI-1 also binds PtdIns(3,5)P2. By homology modelling we earlier identified 24 evolutionarily highly conserved amino acids that cluster at two opposite sites of the open Velcro arranged WIPI-1 β-propeller.Results: By alanine scanning mutagenesis of 24 conserved residues in human WIPI-1 we define the PtdIns-binding site of human WIPI-1 to critically include S203, S205, G208, T209, R212, R226, R227, G228, S251, T255, H257. These amino acids confer PtdIns(3)P or PtdIns(3,5)P2 binding. In general, WIPI-1 mutants unable to bind PtdIns(3)P/PtdIns (3,5)P2 lost their potential to localize at autophagosomal membranes, but WIPI-1 mutants that retained PtdIns(3)P/ PtdIns(3,5)P2 binding localized at Atg12-positive phagophores upon mTORC1 inhibition. Both, downregulation of mTOR by siRNA or cellular PtdIns(3)P elevation upon PIKfyve inhibition by YM201636 significantly increased the localization of WIPI-1 at autophagosomal membranes. Further, we identified regulatory amino acids that influence the membrane recruitment of WIPI-1. Exceptional, WIPI-1 R110A localization at Atg12-positive membranes was independent of autophagy stimulation and insensitive to wortmannin. R112A and H185A mutants were unable to bind PtdIns(3)P/PtdIns(3,5)P2 but localized at autophagosomal membranes, although in a significant reduced number of cells when compared to wild-type WIPI-1.Conclusions: We identified amino acids of the WIPI-1 β-propeller that confer PtdIns(3)P or PtdIns(3,5)P2 binding (S203, S205, G208, T209, R212, R226, R227, G228, S251, T255, H257), and that regulate the localization at autophagosomal membranes (R110, R112, H185) downstream of mTORC1 inhibition.