CheekAge: a next-generation buccal epigenetic aging clock associated with lifestyle and health.

Epigenetic aging clocks are computational models that predict age using DNA methylation information. Initially, first-generation clocks were developed to make predictions using CpGs that change with age. Over time, next-generation clocks were created using CpGs that relate to both age and health. Since existing next-generation clocks were constructed in blood, we sought to develop a next-generation clock optimized for prediction in cheek swabs, which are non-invasive and easy to collect. To do this, we collected MethylationEPIC data as well as lifestyle and health information from 8045 diverse adults. Using a novel simulated annealing approach that allowed us to incorporate lifestyle and health factors into training as well as a combination of CpG filtering, CpG clustering, and clock ensembling, we constructed CheekAge, an epigenetic aging clock that has a strong correlation with age, displays high test-retest reproducibility across replicates, and significantly associates with a plethora of lifestyle and health factors, such as BMI, smoking status, and alcohol intake. We validated CheekAge in an internal dataset and multiple publicly available datasets, including samples from patients with progeria or meningioma. In addition to exploring the underlying biology of the data and clock, we provide a free online tool that allows users to mine our methylomic data and predict epigenetic age.

Morphological diversification and functional maturation of human astrocytes in glia-enriched cortical organoid transplanted in mouse brain.

Astrocytes, the most abundant glial cell type in the brain, are underrepresented in traditional cortical organoid models due to the delayed onset of cortical gliogenesis. Here we introduce a new glia-enriched cortical organoid model that exhibits accelerated astrogliogenesis. We demonstrated that induction of a gliogenic switch in a subset of progenitors enabled the rapid derivation of astroglial cells, which account for 25-31% of the cell population within 8-10 weeks of differentiation. Intracerebral transplantation of these organoids reliably generated a diverse repertoire of cortical neurons and anatomical subclasses of human astrocytes. Spatial transcriptome profiling identified layer-specific expression patterns among distinct subclasses of astrocytes within organoid transplants. Using an in vivo acute neuroinflammation model, we identified a subpopulation of astrocytes that rapidly activates pro-inflammatory pathways upon cytokine stimulation. Additionally, we demonstrated that CD38 signaling has a crucial role in mediating metabolic and mitochondrial stress in reactive astrocytes. This model provides a robust platform for investigating human astrocyte function.

Transgenerational inheritance of acquired epigenetic signatures at CpG islands in mice.

Transgenerational epigenetic inheritance in mammals remains a debated subject. Here, we demonstrate that DNA methylation of promoter-associated CpG islands (CGIs) can be transmitted from parents to their offspring in mice. We generated DNA methylation-edited mouse embryonic stem cells (ESCs), in which CGIs of two metabolism-related genes, the Ankyrin repeat domain 26 and the low-density lipoprotein receptor, were specifically methylated and silenced. DNA methylation-edited mice generated by microinjection of the methylated ESCs exhibited abnormal metabolic phenotypes. Acquired methylation of the targeted CGI and the phenotypic traits were maintained and transmitted across multiple generations. The heritable CGI methylation was subjected to reprogramming in parental PGCs and subsequently reestablished in the next generation at post-implantation stages. These observations provide a concrete step toward demonstrating transgenerational epigenetic inheritance in mammals, which may have implications in our understanding of evolutionary biology as well as the etiology, diagnosis, and prevention of non-genetically inherited human diseases.

Telomere-to-mitochondria signalling by ZBP1 mediates replicative crisis.

Cancers arise through the accumulation of genetic and epigenetic alterations that enable cells to evade telomere-based proliferative barriers and achieve immortality. One such barrier is replicative crisis-an autophagy-dependent program that eliminates checkpoint-deficient cells with unstable telomeres and other cancer-relevant chromosomal aberrations1,2. However, little is known about the molecular events that regulate the onset of this important tumour-suppressive barrier. Here we identified the innate immune sensor Z-DNA binding protein 1 (ZBP1) as a regulator of the crisis program. A crisis-associated isoform of ZBP1 is induced by the cGAS-STING DNA-sensing pathway, but reaches full activation only when associated with telomeric-repeat-containing RNA (TERRA) transcripts that are synthesized from dysfunctional telomeres. TERRA-bound ZBP1 oligomerizes into filaments on the outer mitochondrial membrane of a subset of mitochondria, where it activates the innate immune adapter protein mitochondrial antiviral-signalling protein (MAVS). We propose that these oligomerization properties of ZBP1 serve as a signal amplification mechanism, where few TERRA-ZBP1 interactions are sufficient to launch a detrimental MAVS-dependent interferon response. Our study reveals a mechanism for telomere-mediated tumour suppression, whereby dysfunctional telomeres activate innate immune responses through mitochondrial TERRA-ZBP1 complexes to eliminate cells destined for neoplastic transformation.

Profiling mouse brown and white adipocytes to identify metabolically relevant small ORFs and functional microproteins.

Microproteins (MPs) are a potentially rich source of uncharacterized metabolic regulators. Here, we use ribosome profiling (Ribo-seq) to curate 3,877 unannotated MP-encoding small ORFs (smORFs) in primary brown, white, and beige mouse adipocytes. Of these, we validated 85 MPs by proteomics, including 33 circulating MPs in mouse plasma. Analyses of MP-encoding mRNAs under different physiological conditions (high-fat diet) revealed that numerous MPs are regulated in adipose tissue in vivo and are co-expressed with established metabolic genes. Furthermore, Ribo-seq provided evidence for the translation of Gm8773, which encodes a secreted MP that is homologous to human and chicken FAM237B. Gm8773 is highly expressed in the arcuate nucleus of the hypothalamus, and intracerebroventricular administration of recombinant mFAM237B showed orexigenic activity in obese mice. Together, these data highlight the value of this adipocyte MP database in identifying MPs with roles in fundamental metabolic and physiological processes such as feeding.

Aging compromises human islet beta cell function and identity by decreasing transcription factor activity and inducing ER stress.

Pancreatic islet beta cells are essential for maintaining glucose homeostasis. To understand the impact of aging on beta cells, we performed meta-analysis of single-cell RNA sequencing datasets, transcription factor (TF) regulon analysis, high-resolution confocal microscopy, and measured insulin secretion from nondiabetic donors spanning most of the human life span. This revealed the range of molecular and functional changes that occur during beta cell aging, including the transcriptional deregulation that associates with cellular immaturity and reorganization of beta cell TF networks, increased gene transcription rates, and reduced glucose-stimulated insulin release. These alterations associate with activation of endoplasmic reticulum (ER) stress and autophagy pathways. We propose that a chronic state of ER stress undermines old beta cell structure function to increase the risk of beta cell failure and type 2 diabetes onset as humans age.

A set of common buccal CpGs that predict epigenetic age and associate with lifespan-regulating genes.

Epigenetic aging clocks are computational models that use DNA methylation sites to predict age. Since cheek swabs are non-invasive and painless, collecting DNA from buccal tissue is highly desirable. Here, we review 11 existing clocks that have been applied to buccal tissue. Two of these were exclusively trained on adults and, while moderately accurate, have not been used to capture health-relevant differences in epigenetic age. Using 130 common CpGs utilized by two or more existing buccal clocks, we generate a proof-of-concept predictor in an adult methylomic dataset. In addition to accurately estimating age (r = 0.95 and mean absolute error = 3.88 years), this clock predicted that Down syndrome subjects were significantly older relative to controls. A literature and database review of CpG-associated genes identified numerous genes (e.g., CLOCK, ELOVL2, and VGF) and molecules (e.g., alpha-linolenic acid, glycine, and spermidine) reported to influence lifespan and/or age-related disease in model organisms.

A neurogenetic mechanism of experience-dependent suppression of aggression.

Aggression is an ethologically important social behavior, but excessive aggression can be detrimental to fitness. Social experiences among conspecific individuals reduce aggression in many species, the mechanism of which is largely unknown. We found that loss-of-function mutation of nervy (nvy), a Drosophila homolog of vertebrate myeloid translocation genes (MTGs), increased aggressiveness only in socially experienced flies and that this could be reversed by neuronal expression of human MTGs. A subpopulation of octopaminergic/tyraminergic neurons labeled by nvy was specifically required for such social experience-dependent suppression of aggression, in both males and females. Cell type-specific transcriptomic analysis of these neurons revealed aggression-controlling genes that are likely downstream of nvy. Our results illustrate both genetic and neuronal mechanisms by which the nervous system suppresses aggression in a social experience-dependent manner, a poorly understood process that is considered important for maintaining the fitness of animals.

The TAM receptor tyrosine kinases Axl and Mer drive the maintenance of highly phagocytic macrophages.

Many apoptotic thymocytes are generated during the course of T cell selection in the thymus, yet the machinery through which these dead cells are recognized and phagocytically cleared is incompletely understood. We found that the TAM receptor tyrosine kinases Axl and Mer, which are co-expressed by a specialized set of phagocytic thymic macrophages, are essential components of this machinery. Mutant mice lacking Axl and Mer exhibited a marked accumulation of apoptotic cells during the time that autoreactive and nonreactive thymocytes normally die. Unexpectedly, these double mutants also displayed a profound deficit in the total number of highly phagocytic macrophages in the thymus, and concomitantly exhibited diminished expression of TIM-4, CD163, and other non-TAM phagocytic engulfment systems in the macrophages that remained. Importantly, these previously unrecognized deficits were not confined to the thymus, as they were also evident in the spleen and bone marrow. They had pleiotropic consequences for the double mutants, also previously unrecognized, which included dysregulation of hemoglobin turnover and iron metabolism leading to anemia.

An integrative machine-learning meta-analysis of high-throughput omics data identifies age-specific hallmarks of Alzheimer's disease.

Alzheimer's disease (AD) is an incredibly complex and presently incurable age-related brain disorder. To better understand this debilitating disease, we collated and performed a meta-analysis on publicly available RNA-Seq, microarray, proteomics, and microRNA samples derived from AD patients and non-AD controls. 4089 samples originating from brain tissues and blood remained after applying quality filters. Since disease progression in AD correlates with age, we stratified this large dataset into three different age groups: < 75 years, 75-84 years, and ≥ 85 years. The RNA-Seq, microarray, and proteomics datasets were then combined into different integrated datasets. Ensemble machine learning was employed to identify genes and proteins that can accurately classify samples as either AD or control. These predictive inputs were then subjected to network-based enrichment analyses. The ability of genes/proteins associated with different pathways in the Molecular Signatures Database to diagnose AD was also tested. We separately identified microRNAs that can be used to make an AD diagnosis and subjected the predicted gene targets of the most predictive microRNAs to an enrichment analysis. The following key themes emerged from our machine learning and bioinformatics analyses: cell death, cellular senescence, energy metabolism, genomic integrity, glia, immune system, metal ion homeostasis, oxidative stress, proteostasis, and synaptic function. Many of the results demonstrated unique age-specificity. For example, terms highlighting cellular senescence only emerged in the earliest and intermediate age ranges while the majority of results relevant to cell death appeared in the youngest patients. Existing literature corroborates the importance of these hallmarks in AD.

Aberrant astrocyte protein secretion contributes to altered neuronal development in multiple models of neurodevelopmental disorders.

Astrocytes negatively impact neuronal development in many models of neurodevelopmental disorders (NDs); however, how they do this, and if mechanisms are shared across disorders, is not known. In this study, we developed a cell culture system to ask how astrocyte protein secretion and gene expression change in three mouse models of genetic NDs (Rett, Fragile X and Down syndromes). ND astrocytes increase release of Igfbp2, a secreted inhibitor of insulin-like growth factor (IGF). IGF rescues neuronal deficits in many NDs, and we found that blocking Igfbp2 partially rescues inhibitory effects of Rett syndrome astrocytes, suggesting that increased astrocyte Igfbp2 contributes to decreased IGF signaling in NDs. We identified that increased BMP signaling is upstream of protein secretion changes, including Igfbp2, and blocking BMP signaling in Fragile X and Rett syndrome astrocytes reverses inhibitory effects on neurite outgrowth. This work provides a resource of astrocyte-secreted proteins in health and ND models and identifies novel targets for intervention in diverse NDs.

Reduced synaptic activity and dysregulated extracellular matrix pathways in midbrain neurons from Parkinson's disease patients.

Several mutations that cause Parkinson's disease (PD) have been identified over the past decade. These account for 15-25% of PD cases; the rest of the cases are considered sporadic. Currently, it is accepted that PD is not a single monolithic disease but rather a constellation of diseases with some common phenotypes. While rodent models exist for some of the PD-causing mutations, research on the sporadic forms of PD is lagging due to a lack of cellular models. In our study, we differentiated PD patient-derived dopaminergic (DA) neurons from the induced pluripotent stem cells (iPSCs) of several PD-causing mutations as well as from sporadic PD patients. Strikingly, we observed a common neurophysiological phenotype: neurons derived from PD patients had a severe reduction in the rate of synaptic currents compared to those derived from healthy controls. While the relationship between mutations in genes such as the SNCA and LRRK2 and a reduction in synaptic transmission has been investigated before, here we show evidence that the pathogenesis of the synapses in neurons is a general phenotype in PD. Analysis of RNA sequencing results displayed changes in gene expression in different synaptic mechanisms as well as other affected pathways such as extracellular matrix-related pathways. Some of these dysregulated pathways are common to all PD patients (monogenic or idiopathic). Our data, therefore, show changes that are central and convergent to PD and suggest a strong involvement of the tetra-partite synapse in PD pathophysiology.

Human age reversal: Fact or fiction?

Although chronological age correlates with various age-related diseases and conditions, it does not adequately reflect an individual's functional capacity, well-being, or mortality risk. In contrast, biological age provides information about overall health and indicates how rapidly or slowly a person is aging. Estimates of biological age are thought to be provided by aging clocks, which are computational models (e.g., elastic net) that use a set of inputs (e.g., DNA methylation sites) to make a prediction. In the past decade, aging clock studies have shown that several age-related diseases, social variables, and mental health conditions associate with an increase in predicted biological age relative to chronological age. This phenomenon of age acceleration is linked to a higher risk of premature mortality. More recent research has demonstrated that predicted biological age is sensitive to specific interventions. Human trials have reported that caloric restriction, a plant-based diet, lifestyle changes involving exercise, a drug regime including metformin, and vitamin D3 supplementation are all capable of slowing down or reversing an aging clock. Non-interventional studies have connected high-quality sleep, physical activity, a healthy diet, and other factors to age deceleration. Specific molecules have been associated with the reduction or reversal of predicted biological age, such as the antihypertensive drug doxazosin or the metabolite alpha-ketoglutarate. Although rigorous clinical trials are needed to validate these initial findings, existing data suggest that aging clocks are malleable in humans. Additional research is warranted to better understand these computational models and the clinical significance of lowering or reversing their outputs.

Wiskott-Aldrich syndrome protein forms nuclear condensates and regulates alternative splicing.

The diverse functions of WASP, the deficiency of which causes Wiskott-Aldrich syndrome (WAS), remain poorly defined. We generated three isogenic WAS models using patient induced pluripotent stem cells and genome editing. These models recapitulated WAS phenotypes and revealed that WASP deficiency causes an upregulation of numerous RNA splicing factors and widespread altered splicing. Loss of WASP binding to splicing factor gene promoters frequently leads to aberrant epigenetic activation. WASP interacts with dozens of nuclear speckle constituents and constrains SRSF2 mobility. Using an optogenetic system, we showed that WASP forms phase-separated condensates that encompasses SRSF2, nascent RNA and active Pol II. The role of WASP in gene body condensates is corroborated by ChIPseq and RIPseq. Together our data reveal that WASP is a nexus regulator of RNA splicing that controls the transcription of splicing factors epigenetically and the dynamics of the splicing machinery through liquid-liquid phase separation.

Glucocorticoid signaling and regulatory T cells cooperate to maintain the hair-follicle stem-cell niche.

Maintenance of tissue homeostasis is dependent on the communication between stem cells and supporting cells in the same niche. Regulatory T cells (Treg cells) are emerging as a critical component of the stem-cell niche for supporting their differentiation. How Treg cells sense dynamic signals in this microenvironment and communicate with stem cells is mostly unknown. In the present study, by using hair follicles (HFs) to study Treg cell-stem cell crosstalk, we show an unrecognized function of the steroid hormone glucocorticoid in instructing skin-resident Treg cells to facilitate HF stem-cell (HFSC) activation and HF regeneration. Ablation of the glucocorticoid receptor (GR) in Treg cells blocks hair regeneration without affecting immune homeostasis. Mechanistically, GR and Foxp3 cooperate in Treg cells to induce transforming growth factor ß3 (TGF-ß3), which activates Smad2/3 in HFSCs and facilitates HFSC proliferation. The present study identifies crosstalk between Treg cells and HFSCs mediated by the GR-TGF-ß3 axis, highlighting a possible means of manipulating Treg cells to support tissue regeneration.

In vivo partial cellular reprogramming enhances liver plasticity and regeneration.

Mammals have limited regenerative capacity, whereas some vertebrates, like fish and salamanders, are able to regenerate their organs efficiently. The regeneration in these species depends on cell dedifferentiation followed by proliferation. We generate a mouse model that enables the inducible expression of the four Yamanaka factors (Oct-3/4, Sox2, Klf4, and c-Myc, or 4F) specifically in hepatocytes. Transient in vivo 4F expression induces partial reprogramming of adult hepatocytes to a progenitor state and concomitantly increases cell proliferation. This is indicated by reduced expression of differentiated hepatic-lineage markers, an increase in markers of proliferation and chromatin modifiers, global changes in DNA accessibility, and an acquisition of liver stem and progenitor cell markers. Functionally, short-term expression of 4F enhances liver regenerative capacity through topoisomerase2-mediated partial reprogramming. Our results reveal that liver-specific 4F expression in vivo induces cellular plasticity and counteracts liver failure, suggesting that partial reprogramming may represent an avenue for enhancing tissue regeneration.

Chronic cortisol differentially impacts stem cell-derived astrocytes from major depressive disorder patients.

Major depressive disorder (MDD) is a prevalent psychiatric disorder, and exposure to stress is a robust risk factor for MDD. Clinical data and rodent models have indicated the negative impact of chronic exposure to stress-induced hormones like cortisol on brain volume, memory, and cell metabolism. However, the cellular and transcriptomic changes that occur in the brain after prolonged exposure to cortisol are less understood. Furthermore, the astrocyte-specific contribution to cortisol-induced neuropathology remains understudied. Here, we have developed an in vitro model of "chronic stress" using human induced pluripotent stem cell (iPSC)-derived astrocytes treated with cortisol for 7 days. Whole transcriptome sequencing reveals differentially expressed genes (DEGs) uniquely regulated in chronic cortisol compared to acute cortisol treatment. Utilizing this paradigm, we examined the stress response transcriptome of astrocytes generated from MDD patient iPSCs. The MDD-specific DEGs are related to GPCR ligand binding, synaptic signaling, and ion homeostasis. Together, these data highlight the unique role astrocytes play in the central nervous system and present interesting genes for future study into the relationship between chronic stress and MDD.

Activity-dependent modulation of synapse-regulating genes in astrocytes.

Astrocytes regulate the formation and function of neuronal synapses via multiple signals; however, what controls regional and temporal expression of these signals during development is unknown. We determined the expression profile of astrocyte synapse-regulating genes in the developing mouse visual cortex, identifying astrocyte signals that show differential temporal and layer-enriched expression. These patterns are not intrinsic to astrocytes, but regulated by visually evoked neuronal activity, as they are absent in mice lacking glutamate release from thalamocortical terminals. Consequently, synapses remain immature. Expression of synapse-regulating genes and synaptic development is also altered when astrocyte signaling is blunted by diminishing calcium release from astrocyte stores. Single-nucleus RNA sequencing identified groups of astrocytic genes regulated by neuronal and astrocyte activity, and a cassette of genes that show layer-specific enrichment. Thus, the development of cortical circuits requires coordinated signaling between astrocytes and neurons, highlighting astrocytes as a target to manipulate in neurodevelopmental disorders.