A platform for rapid exploration of aging and diseases in a naturally short-lived vertebrate.

VIDEO ABSTRACT: Aging is a complex process that affects multiple organs. Modeling aging and age-related diseases in the lab is challenging because classical vertebrate models have relatively long lifespans. Here, we develop the first platform for rapid exploration of age-dependent traits and diseases in vertebrates, using the naturally short-lived African turquoise killifish. We provide an integrative genomic and genome-editing toolkit in this organism using our de-novo-assembled genome and the CRISPR/Cas9 technology. We mutate many genes encompassing the hallmarks of aging, and for a subset, we produce stable lines within 2-3 months. As a proof of principle, we show that fish deficient for the protein subunit of telomerase exhibit the fastest onset of telomere-related pathologies among vertebrates. We further demonstrate the feasibility of creating specific genetic variants. This genome-to-phenotype platform represents a unique resource for studying vertebrate aging and disease in a high-throughput manner and for investigating candidates arising from human genome-wide studies.

The African Turquoise Killifish Genome Provides Insights into Evolution and Genetic Architecture of Lifespan.

Lifespan is a remarkably diverse trait ranging from a few days to several hundred years in nature, but the mechanisms underlying the evolution of lifespan differences remain elusive. Here we de novo assemble a reference genome for the naturally short-lived African turquoise killifish, providing a unique resource for comparative and experimental genomics. The identification of genes under positive selection in this fish reveals potential candidates to explain its compressed lifespan. Several aging genes are under positive selection in this short-lived fish and long-lived species, raising the intriguing possibility that the same gene could underlie evolution of both compressed and extended lifespans. Comparative genomics and linkage analysis identify candidate genes associated with lifespan differences between various turquoise killifish strains. Remarkably, these genes are clustered on the sex chromosome, suggesting that short lifespan might have co-evolved with sex determination. Our study provides insights into the evolutionary forces that shape lifespan in nature.

H3K4me3 breadth is linked to cell identity and transcriptional consistency.

Trimethylation of histone H3 at lysine 4 (H3K4me3) is a chromatin modification known to mark the transcription start sites of active genes. Here, we show that H3K4me3 domains that spread more broadly over genes in a given cell type preferentially mark genes that are essential for the identity and function of that cell type. Using the broadest H3K4me3 domains as a discovery tool in neural progenitor cells, we identify novel regulators of these cells. Machine learning models reveal that the broadest H3K4me3 domains represent a distinct entity, characterized by increased marks of elongation. The broadest H3K4me3 domains also have more paused polymerase at their promoters, suggesting a unique transcriptional output. Indeed, genes marked by the broadest H3K4me3 domains exhibit enhanced transcriptional consistency and [corrected] increased transcriptional levels, and perturbation of H3K4me3 breadth leads to changes in transcriptional consistency. Thus, H3K4me3 breadth contains information that could ensure transcriptional precision at key cell identity/function genes.

Epigenetic regulation of ageing: linking environmental inputs to genomic stability.

Ageing is affected by both genetic and non-genetic factors. Here, we review the chromatin-based epigenetic changes that occur during ageing, the role of chromatin modifiers in modulating lifespan and the importance of epigenetic signatures as biomarkers of ageing. We also discuss how epigenome remodelling by environmental stimuli affects several aspects of transcription and genomic stability, with important consequences for longevity, and outline epigenetic differences between the 'mortal soma' and the 'immortal germ line'. Finally, we discuss the inheritance of characteristics of ageing and potential chromatin-based strategies to delay or reverse hallmarks of ageing or age-related diseases.

An eQTL-based approach reveals candidate regulators of LINE-1 RNA levels in lymphoblastoid cells.

Long interspersed element 1 (LINE-1; L1) are a family of transposons that occupy ~17% of the human genome. Though a small number of L1 copies remain capable of autonomous transposition, the overwhelming majority of copies are degenerate and immobile. Nevertheless, both mobile and immobile L1s can exert pleiotropic effects (promoting genome instability, inflammation, or cellular senescence) on their hosts, and L1's contributions to aging and aging diseases is an area of active research. However, because of the cell type-specific nature of transposon control, the catalogue of L1 regulators remains incomplete. Here, we employ an eQTL approach leveraging transcriptomic and genomic data from the GEUVADIS and 1000Genomes projects to computationally identify new candidate regulators of L1 RNA levels in lymphoblastoid cell lines. To cement the role of candidate genes in L1 regulation, we experimentally modulate the levels of top candidates in vitro, including IL16, STARD5, HSD17B12, and RNF5, and assess changes in TE family expression by Gene Set Enrichment Analysis (GSEA). Remarkably, we observe subtle but widespread upregulation of TE family expression following IL16 and STARD5 overexpression. Moreover, a short-term 24-hour exposure to recombinant human IL16 was sufficient to transiently induce subtle, but widespread, upregulation of L1 subfamilies. Finally, we find that many L1 expression-associated genetic variants are co-associated with aging traits across genome-wide association study databases. Our results expand the catalogue of genes implicated in L1 RNA control and further suggest that L1-derived RNA contributes to aging processes. Given the ever-increasing availability of paired genomic and transcriptomic data, we anticipate this new approach to be a starting point for more comprehensive computational scans for regulators of transposon RNA levels.

Dynamic regulation of gonadal transposon control across the lifespan of the naturally short-lived African turquoise killifish.

Although germline cells are considered to be functionally "immortal," both the germline and supporting somatic cells in the gonad within an organism experience aging. With increased age at parenthood, the age-related decline in reproductive success has become an important biological issue for an aging population. However, molecular mechanisms underlying reproductive aging across sexes in vertebrates remain poorly understood. To decipher molecular drivers of vertebrate gonadal aging across sexes, we perform longitudinal characterization of the gonadal transcriptome throughout the lifespan in the naturally short-lived African turquoise killifish (Nothobranchius furzeri). By combining mRNA-seq and small RNA-seq from 26 individuals, we characterize the aging gonads of young-adult, middle-aged, and old female and male fish. We analyze changes in transcriptional patterns of genes, transposable elements (TEs), and piRNAs. We find that testes seem to undergo only marginal changes during aging. In contrast, in middle-aged ovaries, the time point associated with peak female fertility in this strain, PIWI pathway components are transiently down-regulated, TE transcription is elevated, and piRNA levels generally decrease, suggesting that egg quality may already be declining at middle-age. Furthermore, we show that piRNA ping-pong biogenesis declines steadily with age in ovaries, whereas it is maintained in aging testes. To our knowledge, this data set represents the most comprehensive transcriptomic data set for vertebrate gonadal aging. This resource also highlights important pathways that are regulated during reproductive aging in either ovaries or testes, which could ultimately be leveraged to help restore aspects of youthful reproductive function.

The fidelity of transcription in human cells.

To determine the error rate of transcription in human cells, we analyzed the transcriptome of H1 human embryonic stem cells with a circle-sequencing approach that allows for high-fidelity sequencing of the transcriptome. These experiments identified approximately 100,000 errors distributed over every major RNA species in human cells. Our results indicate that different RNA species display different error rates, suggesting that human cells prioritize the fidelity of some RNAs over others. Cross-referencing the errors that we detected with various genetic and epigenetic features of the human genome revealed that the in vivo error rate in human cells changes along the length of a transcript and is further modified by genetic context, repetitive elements, epigenetic markers, and the speed of transcription. Our experiments further suggest that BRCA1, a DNA repair protein implicated in breast cancer, has a previously unknown role in the suppression of transcription errors. Finally, we analyzed the distribution of transcription errors in multiple tissues of a new mouse model and found that they occur preferentially in neurons, compared to other cell types. These observations lend additional weight to the idea that transcription errors play a key role in the progression of various neurological disorders, including Alzheimer's disease.

Heterogeneity in old fibroblasts is linked to variability in reprogramming and wound healing.

Age-associated chronic inflammation (inflammageing) is a central hallmark of ageing1, but its influence on specific cells remains largely unknown. Fibroblasts are present in most tissues and contribute to wound healing2,3. They are also the most widely used cell type for reprogramming to induced pluripotent stem (iPS) cells, a process that has implications for regenerative medicine and rejuvenation strategies4. Here we show that fibroblast cultures from old mice secrete inflammatory cytokines and exhibit increased variability in the efficiency of iPS cell reprogramming between mice. Variability between individuals is emerging as a feature of old age5-8, but the underlying mechanisms remain unknown. To identify drivers of this variability, we performed multi-omics profiling of fibroblast cultures from young and old mice that have different reprogramming efficiencies. This approach revealed that fibroblast cultures from old mice contain 'activated fibroblasts' that secrete inflammatory cytokines, and that the proportion of activated fibroblasts in a culture correlates with the reprogramming efficiency of that culture. Experiments in which conditioned medium was swapped between cultures showed that extrinsic factors secreted by activated fibroblasts underlie part of the variability between mice in reprogramming efficiency, and we have identified inflammatory cytokines, including TNF, as key contributors. Notably, old mice also exhibited variability in wound healing rate in vivo. Single-cell RNA-sequencing analysis identified distinct subpopulations of fibroblasts with different cytokine expression and signalling in the wounds of old mice with slow versus fast healing rates. Hence, a shift in fibroblast composition, and the ratio of inflammatory cytokines that they secrete, may drive the variability between mice in reprogramming in vitro and influence wound healing rate in vivo. This variability may reflect distinct stochastic ageing trajectories between individuals, and could help in developing personalized strategies to improve iPS cell generation and wound healing in elderly individuals.

Microglia undergo sex-dimorphic transcriptional and metabolic rewiring during aging.

Microglia, the brain's resident macrophages, maintain brain homeostasis and respond to injury and infection. During aging they undergo functional changes, but the underlying mechanisms and their contributions to neuroprotection versus neurodegeneration are unclear. Previous studies suggested that microglia are sex dimorphic, so we compared microglial aging in mice of both sexes. RNA-sequencing of hippocampal microglia revealed more aging-associated changes in female microglia than male microglia, and more sex differences in old microglia than young microglia. Pathway analyses and subsequent validation assays revealed a stronger AKT-mTOR-HIF1α-driven shift to glycolysis among old female microglia and indicated that C3a production and detection was elevated in old microglia, especially in females. Recombinant C3a induced AKT-mTOR-HIF1α signaling and increased the glycolytic and phagocytic activity of young microglia. Single cell analyses attributed the aging-associated sex dimorphism to more abundant disease-associated microglia (DAM) in old female mice than old male mice, and evaluation of an Alzheimer's Disease mouse model revealed that the metabolic and complement changes are also apparent in the context of neurodegenerative disease and are strongest in the neuroprotective DAM2 subset. Collectively, our data implicate autocrine C3a-C3aR signaling in metabolic reprogramming of microglia to neuroprotective DAM during aging, especially in females, and also in Alzheimer's Disease.

Transposable elements, circular RNAs and mitochondrial transcription in age-related genomic regulation.

Our understanding of the molecular regulation of aging and age-related diseases is still in its infancy, requiring in-depth characterization of the molecular landscape shaping these complex phenotypes. Emerging classes of molecules with promise as aging modulators include transposable elements, circRNAs and the mitochondrial transcriptome. Analytical complexity means that these molecules are often overlooked, even though they exhibit strong associations with aging and, in some cases, may directly contribute to its progress. Here, we review the links between these novel factors and age-related phenotypes, and we suggest tools that can be easily incorporated into existing pipelines to better understand the aging process.

Sex as a Biological Variable in Nutrition Research: From Human Studies to Animal Models.

Biological sex is a fundamental source of phenotypic variability across species. Males and females have different nutritional needs and exhibit differences in nutrient digestion and utilization, leading to different health outcomes throughout life. With personalized nutrition gaining popularity in scientific research and clinical practice, it is important to understand the fundamentals of sex differences in nutrition research. Here, we review key studies that investigate sex dimorphism in nutrition research: sex differences in nutrient intake and metabolism, sex-dimorphic response in nutrient-restricted conditions, and sex differences in diet and gut microbiome interactions. Within each area above, factors from sex chromosomes, sex hormones, and sex-specific loci are highlighted.

Endogenous Retroviral Elements Generate Pathologic Neutrophils in Pulmonary Arterial Hypertension.

Rationale: The role of neutrophils and their extracellular vesicles (EVs) in the pathogenesis of pulmonary arterial hypertension is unclear. Objectives: To relate functional abnormalities in pulmonary arterial hypertension neutrophils and their EVs to mechanisms uncovered by proteomic and transcriptomic profiling. Methods: Production of elastase, release of extracellular traps, adhesion, and migration were assessed in neutrophils from patients with pulmonary arterial hypertension and control subjects. Proteomic analyses were applied to explain functional perturbations, and transcriptomic data were used to find underlying mechanisms. CD66b-specific neutrophil EVs were isolated from plasma of patients with pulmonary arterial hypertension, and we determined whether they produce pulmonary hypertension in mice. Measurements and Main Results: Neutrophils from patients with pulmonary arterial hypertension produce and release increased neutrophil elastase, associated with enhanced extracellular traps. They exhibit reduced migration and increased adhesion attributed to elevated ß1-integrin and vinculin identified by proteomic analysis and previously linked to an antiviral response. This was substantiated by a transcriptomic IFN signature that we related to an increase in human endogenous retrovirus K envelope protein. Transfection of human endogenous retrovirus K envelope in a neutrophil cell line (HL-60) increases neutrophil elastase and IFN genes, whereas vinculin is increased by human endogenous retrovirus K deoxyuridine triphosphate diphosphatase that is elevated in patient plasma. Neutrophil EVs from patient plasma contain increased neutrophil elastase and human endogenous retrovirus K envelope and induce pulmonary hypertension in mice, mitigated by elafin, an elastase inhibitor. Conclusions: Elevated human endogenous retroviral elements and elastase link a neutrophil innate immune response to pulmonary arterial hypertension.

High telomerase is a hallmark of undifferentiated spermatogonia and is required for maintenance of male germline stem cells.

Telomerase inactivation causes loss of the male germline in worms, fish, and mice, indicating a conserved dependence on telomere maintenance in this cell lineage. Here, using telomerase reverse transcriptase (Tert) reporter mice, we found that very high telomerase expression is a hallmark of undifferentiated spermatogonia, the mitotic population where germline stem cells reside. We exploited these high telomerase levels as a basis for purifying undifferentiated spermatogonia using fluorescence-activated cell sorting. Telomerase levels in undifferentiated spermatogonia and embryonic stem cells are comparable and much greater than in somatic progenitor compartments. Within the germline, we uncovered an unanticipated gradient of telomerase activity that also enables isolation of more mature populations. Transcriptomic comparisons of Tert(High) undifferentiated spermatogonia and Tert(Low) differentiated spermatogonia by RNA sequencing reveals marked differences in cell cycle and key molecular features of each compartment. Transplantation studies show that germline stem cell activity is confined to the Tert(High) cKit(-) population. Telomere shortening in telomerase knockout strains causes depletion of undifferentiated spermatogonia and eventual loss of all germ cells after undifferentiated spermatogonia drop below a critical threshold. These data reveal that high telomerase expression is a fundamental characteristic of germline stem cells, thus explaining the broad dependence on telomerase for germline immortality in metazoans.

Remodeling of epigenome and transcriptome landscapes with aging in mice reveals widespread induction of inflammatory responses.

Aging is accompanied by the functional decline of tissues. However, a systematic study of epigenomic and transcriptomic changes across tissues during aging is missing. Here, we generated chromatin maps and transcriptomes from four tissues and one cell type from young, middle-aged, and old mice-yielding 143 high-quality data sets. We focused on chromatin marks linked to gene expression regulation and cell identity: histone H3 trimethylation at lysine 4 (H3K4me3), a mark enriched at promoters, and histone H3 acetylation at lysine 27 (H3K27ac), a mark enriched at active enhancers. Epigenomic and transcriptomic landscapes could easily distinguish between ages, and machine-learning analysis showed that specific epigenomic states could predict transcriptional changes during aging. Analysis of data sets from all tissues identified recurrent age-related chromatin and transcriptional changes in key processes, including the up-regulation of immune system response pathways such as the interferon response. The up-regulation of the interferon response pathway with age was accompanied by increased transcription and chromatin remodeling at specific endogenous retroviral sequences. Pathways misregulated during mouse aging across tissues, notably innate immune pathways, were also misregulated with aging in other vertebrate species-African turquoise killifish, rat, and humans-indicating common signatures of age across species. To date, our data set represents the largest multitissue epigenomic and transcriptomic data set for vertebrate aging. This resource identifies chromatin and transcriptional states that are characteristic of young tissues, which could be leveraged to restore aspects of youthful functionality to old tissues.

MOTS-c: A Mitochondrial-Encoded Regulator of the Nucleus.

Mitochondria are increasingly being recognized as information hubs that sense cellular changes and transmit messages to other cellular components, such as the nucleus, the endoplasmic reticulum (ER), the Golgi apparatus, and lysosomes. Nonetheless, the interaction between mitochondria and the nucleus is of special interest because they both host part of the cellular genome. Thus, the communication between genome-bearing organelles would likely include gene expression regulation. Multiple nuclear-encoded proteins have been known to regulate mitochondrial gene expression. On the contrary, no mitochondrial-encoded factors are known to actively regulate nuclear gene expression. MOTS-c (mitochondrial open reading frame of the 12S ribosomal RNA type-c) is a recently identified peptide encoded within the mitochondrial 12S ribosomal RNA gene that has metabolic functions. Notably, MOTS-c can translocate to the nucleus upon metabolic stress (e.g., glucose restriction and oxidative stress) and directly regulate adaptive nuclear gene expression to promote cellular homeostasis. It is hypothesized that cellular fitness requires the coevolved mitonuclear genomes to coordinate adaptive responses using gene-encoded factors that cross-regulate the opposite genome. This suggests that cellular gene expression requires the bipartite split genomes to operate as a unified system, rather than the nucleus being the sole master regulator.

Widespread sex dimorphism in aging and age-related diseases.

Although aging is a conserved phenomenon across evolutionary distant species, aspects of the aging process have been found to differ between males and females of the same species. Indeed, observations across mammalian studies have revealed the existence of longevity and health disparities between sexes, including in humans (i.e. with a female or male advantage). However, the underlying mechanisms for these sex differences in health and lifespan remain poorly understood, and it is unclear which aspects of this dimorphism stem from hormonal differences (i.e. predominance of estrogens vs. androgens) or from karyotypic differences (i.e. XX vs. XY sex chromosome complement). In this review, we discuss the state of the knowledge in terms of sex dimorphism in various aspects of aging and in human age-related diseases. Where the interplay between sex differences and age-related differences has not been explored fully, we present the state of the field to highlight important future research directions. We also discuss various dietary, drug or genetic interventions that were shown to improve longevity in a sex-dimorphic fashion. Finally, emerging tools and models that can be leveraged to decipher the mechanisms underlying sex differences in aging are also briefly discussed.

Transgenerational epigenetic inheritance of longevity in Caenorhabditis elegans.

Chromatin modifiers regulate lifespan in several organisms, raising the question of whether changes in chromatin states in the parental generation could be incompletely reprogrammed in the next generation and thereby affect the lifespan of descendants. The histone H3 lysine 4 trimethylation (H3K4me3) complex, composed of ASH-2, WDR-5 and the histone methyltransferase SET-2, regulates Caenorhabditis elegans lifespan. Here we show that deficiencies in the H3K4me3 chromatin modifiers ASH-2, WDR-5 or SET-2 in the parental generation extend the lifespan of descendants up until the third generation. The transgenerational inheritance of lifespan extension by members of the ASH-2 complex is dependent on the H3K4me3 demethylase RBR-2, and requires the presence of a functioning germline in the descendants. Transgenerational inheritance of lifespan is specific for the H3K4me3 methylation complex and is associated with epigenetic changes in gene expression. Thus, manipulation of specific chromatin modifiers only in parents can induce an epigenetic memory of longevity in descendants.

Discovery of novel protein partners of the transcription factor FOXL2 provides insights into its physiopathological roles.

FOXL2 transcription factor is responsible for the Blepharophimosis Ptosis Epicantus inversus Syndrome (BPES), a genetic disease involving craniofacial malformations often associated with ovarian failure. Recently, a somatic FOXL2 mutation (p.C134W) has been reported in >95% of adult-type granulosa cell tumors. Here, we have identified 10 novel FOXL2 partners by yeast-two-hybrid screening and co-immunoprecipitation. Most BPES-inducing mutated FOXL2 proteins display aggregation in cultured cells. Here, we show that two of the partners (NR2C1 and GMEB1) can be sequestered in such aggregates. This co-aggregation can contribute to the pathogenesis of FOXL2 mutations. We have also measured the effects of FOXL2 interactants on the transcriptional regulation of a series of target promoters. Some of the partners (CXXC4, CXXC5, BANF1) were able to repress FOXL2 activity indistinctively of the promoter. Interestingly, CREM-τ2α, which acted as a repressor on most promoters, increased wild-type (WT) FOXL2 activity on two promoters (PTGS2 and CYP19A1), but was unable to increase the activity of the oncogenic mutant p.C134W. Conversely, GMEB1, which also acted as a repressor on most promoters and increased WT FOXL2 activity on the Per2 promoter, increased to a greater extent the activity of the p.C134W variant. Interestingly, partners with intrinsic pro-apoptotic effect were able to increase apoptosis induction by WT FOXL2, but not by the p.C134W mutant, whereas partners with an anti-apoptotic effect decreased apoptosis induction by both FOXL2 versions. Altogether, these results suggest that the p.C134W mutated form fails to integrate signals through protein-protein interactions to regulate target promoter subsets and in particular to induce cell death.