Epigenetic signature of human immune aging in the GESTALT study.

Age-associated DNA methylation in blood cells convey information on health status. However, the mechanisms that drive these changes in circulating cells and their relationships to gene regulation are unknown. We identified age-associated DNA methylation sites in six purified blood-borne immune cell types (naive B, naive CD4+ and CD8+ T cells, granulocytes, monocytes, and NK cells) collected from healthy individuals interspersed over a wide age range. Of the thousands of age-associated sites, only 350 sites were differentially methylated in the same direction in all cell types and validated in an independent longitudinal cohort. Genes close to age-associated hypomethylated sites were enriched for collagen biosynthesis and complement cascade pathways, while genes close to hypermethylated sites mapped to neuronal pathways. In silico analyses showed that in most cell types, the age-associated hypo- and hypermethylated sites were enriched for ARNT (HIF1ß) and REST transcription factor (TF) motifs, respectively, which are both master regulators of hypoxia response. To conclude, despite spatial heterogeneity, there is a commonality in the putative regulatory role with respect to TF motifs and histone modifications at and around these sites. These features suggest that DNA methylation changes in healthy aging may be adaptive responses to fluctuations of oxygen availability.

Personality traits are consistently associated with blood mitochondrial DNA copy number estimated from genome sequences in two genetic cohort studies.

Background: Mitochondrial DNA copy number (mtDNAcn) in tissues and blood can be altered in conditions like diabetes and major depression and may play a role in aging and longevity. However, little is known about the association between mtDNAcn and personality traits linked to emotional states, metabolic health, and longevity. This study tests the hypothesis that blood mtDNAcn is related to personality traits and mediates the association between personality and mortality. Methods: We assessed the big five personality domains and facets using the Revised NEO Personality Inventory (NEO-PI-R), assessed depressive symptoms with the Center for Epidemiologic Studies Depression Scale (CES-D), estimated mtDNAcn levels from whole-genome sequencing, and tracked mortality in participants from the Baltimore Longitudinal Study of Aging. Results were replicated in the SardiNIA Project. Results: We found that mtDNAcn was negatively associated with the Neuroticism domain and its facets and positively associated with facets from the other four domains. The direction and size of the effects were replicated in the SardiNIA cohort and were robust to adjustment for potential confounders in both samples. Consistent with the Neuroticism finding, higher depressive symptoms were associated with lower mtDNAcn. Finally, mtDNAcn mediated the association between personality and mortality risk. Conclusions: To our knowledge, this is the first study to show a replicable association between mtDNAcn and personality. Furthermore, the results support our hypothesis that mtDNAcn is a biomarker of the biological process that explains part of the association between personality and mortality. Funding: Support for this work was provided by the Intramural Research Program of the National Institute on Aging (Z01-AG000693, Z01-AG000970, and Z01-AG000949) and the National Institute of Neurological Disorders and Stroke of the National Institutes of Health. AT was also supported by the National Institute on Aging of the National Institutes of Health Grant R01AG068093.

Cells are powered by internal structures called mitochondria which have their own DNA molecules. How many copies of mitochondrial DNA blood cells contain is one aspect of mitochondrial health and is considered to provide a good indication of an individual's ability to convert glucose into energy. Consequently, changes in the amount of mitochondrial DNA in the blood are linked to conditions like diabetes and cancer, and have also been associated with aging and mortality. A set of well-classified personality traits known as 'the Big Five' have also been shown to affect energy levels and the longevity of individuals. However, it remained unclear if there is a relationship between these characteristics and the number of copies of mitochondrial DNA in the blood. To investigate, Oppong et al. used a specialized test to assess the personality traits of participants from two separate cohorts: Baltimore Longitudinal Study of Ageing and the SardiNIA Project. The genomic sequence of each person was then analyzed to calculate the amount of mitochondrial DNA in their blood, and their mortality was recorded based on whether they were alive or dead multiple years later. Oppong et al. found that low levels of mitochondrial DNA were linked with high scores in neuroticism (a trait typically associated with anxiety, depression, and self-doubt). Further statistical tests revealed that mitochondrial DNA levels mediate the relationship between a person's personality and their risk of death. These findings suggest that personality traits impact the number of mitochondrial DNA molecules in a person's blood, which, in turn, influences how long they are likely to live. However, further work is needed to find out what causes this effect.

The Heart's Pacemaker Mimics Brain Cytoarchitecture and Function: Novel Interstitial Cells Expose Complexity of the SAN.

BACKGROUND: The sinoatrial node (SAN) of the heart produces rhythmic action potentials, generated via calcium signaling within and among pacemaker cells. Our previous work has described the SAN as composed of a hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4)-expressing pacemaker cell meshwork, which merges with a network of connexin 43+/F-actin+ cells. It is also known that sympathetic and parasympathetic innervation create an autonomic plexus in the SAN that modulates heart rate and rhythm. However, the anatomical details of the interaction of this plexus with the pacemaker cell meshwork have yet to be described. OBJECTIVES: This study sought to describe the 3-dimensional cytoarchitecture of the mouse SAN, including autonomic innervation, peripheral glial cells, and pacemaker cells. METHODS: The cytoarchitecture of SAN whole-mount preparations was examined by three-dimensional confocal laser-scanning microscopy of triple immunolabeled with combinations of antibodies for HCN4, S100 calcium-binding protein B (S100B), glial fibrillary acidic protein (GFAP), choline acetyltransferase, or vesicular acetylcholine transporter, and tyrosine hydroxylase, and transmission electron microscopy. RESULTS: The SAN exhibited heterogeneous autonomic innervation, which was accompanied by a web of peripheral glial cells and a novel S100B+/GFAP- interstitial cell population, with a unique morphology and a distinct distribution pattern, creating complex interactions with other cell types in the node, particularly with HCN4-expressing cells. Transmission electron microscopy identified a similar population of interstitial cells as telocytes, which appeared to secrete vesicles toward pacemaker cells. Application of S100B to SAN preparations desynchronized Ca2+ signaling in HCN4-expressing cells and increased variability in SAN impulse rate and rhythm. CONCLUSIONS: The autonomic plexus, peripheral glial cell web, and a novel S100B+/GFAP- interstitial cell type embedded within the HCN4+ cell meshwork increase the structural and functional complexity of the SAN and provide a new regulatory pathway of rhythmogenesis.

Male rat leukocyte population dynamics predict a window for intervention in aging.

Many age-associated changes in the human hematopoietic system have been reproduced in murine models; however, such changes have not been as robustly explored in rats despite the fact these larger rodents are more physiologically similar to humans. We examined peripheral blood of male F344 rats ranging from 3 to 27 months of age and found significant age-associated changes with distinct leukocyte population shifts. We report CD25+ CD4+ population frequency is a strong predictor of healthy aging, generate a model using blood parameters, and find rats with blood profiles that diverge from chronologic age indicate debility; thus, assessments of blood composition may be useful for non-lethal disease profiling or as a surrogate measure for efficacy of aging interventions. Importantly, blood parameters and DNA methylation alterations, defined distinct juncture points during aging, supporting a non-linear aging process. Our results suggest these inflection points are important considerations for aging interventions. Overall, we present rat blood aging metrics that can serve as a resource to evaluate health and the effects of interventions in a model system physiologically more reflective of humans.

Our blood contains many types of white blood cells, which play important roles in defending the body against infections and other threats to our health. The number of these cells changes with age, and this in turn contributes to many other alterations that happen in the body as we get older. For example, the immune system generally gets weaker at fighting infections and preventing other cells from developing into cancer. On top of that, the white blood cells themselves can become cancerous, resulting in several types of blood cancer that are more likely to happen in older people. Many previous studies have examined how the number of white blood cells changes with age in humans and mice. However, our understanding of this process in rats is still poor, despite the fact that the way the human body works has more in common with the rat body than the mouse body. Here, Yanai, Dunn et al. have studied samples of blood from rats between three to 27 months old. The experiments found that it is possible to accurately predict the age of healthy rats by measuring the frequency of populations of white blood cells, especially a certain type known as CD25+ CD4+ cells. If the animals had any form of illness, their predicted age deviated from their actual age. Furthermore, while some changes in the blood were gradual and continuous, others displayed distinct shifts when the rats reached specific ages. In the future, these findings may be used as a tool to help researchers diagnose illnesses in rats before the animals develop symptoms, or to more easily establish if a treatment is having a positive effect on the rats' health. The work of Yanai, Dunn et al. also provides new insights into aging that could potentially aid the design of new screening methods to predict cancer and intervene using a model system that is more similar to humans.

DNA methylation signatures reveal that distinct combinations of transcription factors specify human immune cell epigenetic identity.

Epigenetic reprogramming underlies specification of immune cell lineages, but patterns that uniquely define immune cell types and the mechanisms by which they are established remain unclear. Here, we identified lineage-specific DNA methylation signatures of six immune cell types from human peripheral blood and determined their relationship to other epigenetic and transcriptomic patterns. Sites of lineage-specific hypomethylation were associated with distinct combinations of transcription factors in each cell type. By contrast, sites of lineage-specific hypermethylation were restricted mostly to adaptive immune cells. PU.1 binding sites were associated with lineage-specific hypo- and hypermethylation in different cell types, suggesting that it regulates DNA methylation in a context-dependent manner. These observations indicate that innate and adaptive immune lineages are specified by distinct epigenetic mechanisms via combinatorial and context-dependent use of key transcription factors. The cell-specific epigenomics and transcriptional patterns identified serve as a foundation for future studies on immune dysregulation in diseases and aging.

Systematic Identification of circRNAs in Alzheimer's Disease.

Mammalian circRNAs are covalently closed circular RNAs often generated through backsplicing of precursor linear RNAs. Although their functions are largely unknown, they have been found to influence gene expression at different levels and in a wide range of biological processes. Here, we investigated if some circRNAs may be differentially abundant in Alzheimer's Disease (AD). We identified and analyzed publicly available RNA-sequencing data from the frontal lobe, temporal cortex, hippocampus, and plasma samples reported from persons with AD and persons who were cognitively normal, focusing on circRNAs shared across these datasets. We identified an overlap of significantly changed circRNAs among AD individuals in the various brain datasets, including circRNAs originating from genes strongly linked to AD pathology such as DOCK1, NTRK2, APC (implicated in synaptic plasticity and neuronal survival) and DGL1/SAP97, TRAPPC9, and KIF1B (implicated in vesicular traffic). We further predicted the presence of circRNA isoforms in AD using specialized statistical analysis packages to create approximations of entire circRNAs. We propose that the catalog of differentially abundant circRNAs can guide future investigation on the expression and splicing of the host transcripts, as well as the possible roles of these circRNAs in AD pathogenesis.

Skeletal muscle transcriptome in healthy aging.

Age-associated changes in gene expression in skeletal muscle of healthy individuals reflect accumulation of damage and compensatory adaptations to preserve tissue integrity. To characterize these changes, RNA was extracted and sequenced from muscle biopsies collected from 53 healthy individuals (22-83 years old) of the GESTALT study of the National Institute on Aging-NIH. Expression levels of 57,205 protein-coding and non-coding RNAs were studied as a function of aging by linear and negative binomial regression models. From both models, 1134 RNAs changed significantly with age. The most differentially abundant mRNAs encoded proteins implicated in several age-related processes, including cellular senescence, insulin signaling, and myogenesis. Specific mRNA isoforms that changed significantly with age in skeletal muscle were enriched for proteins involved in oxidative phosphorylation and adipogenesis. Our study establishes a detailed framework of the global transcriptome and mRNA isoforms that govern muscle damage and homeostasis with age.

Synchronized Cardiac Impulses Emerge From Heterogeneous Local Calcium Signals Within and Among Cells of Pacemaker Tissue.

OBJECTIVES: This study sought to identify subcellular Ca2+ signals within and among cells comprising the sinoatrial node (SAN) tissue. BACKGROUND: The current paradigm of SAN impulse generation: 1) is that full-scale action potentials (APs) of a common frequency are initiated at 1 site and are conducted within the SAN along smooth isochrones; and 2) does not feature fine details of Ca2+ signaling present in isolated SAN cells, in which small subcellular, subthreshold local Ca2+ releases (LCRs) self-organize to generate cell-wide APs. METHODS: Immunolabeling was combined with a novel technique to detect the occurrence of LCRs and AP-induced Ca2+ transients (APCTs) in individual pixels (chronopix) across the entire mouse SAN images. RESULTS: At high magnification, Ca2+ signals appeared markedly heterogeneous in space, amplitude, frequency, and phase among cells comprising an HCN4+/CX43- cell meshwork. The signaling exhibited several distinguishable patterns of LCR/APCT interactions within and among cells. Rhythmic APCTs that were apparently conducted within the meshwork were transferred to a truly conducting HCN4-/CX43+ network of striated cells via narrow functional interfaces where different cell types intertwine, that is, the SAN anatomic/functional unit. At low magnification, the earliest APCT of each cycle occurred within a small area of the HCN4 meshwork, and subsequent APCT appearance throughout SAN pixels was discontinuous and asynchronous. CONCLUSIONS: The study has discovered a novel, microscopic Ca2+ signaling paradigm of SAN operation that has escaped detection using low-resolution, macroscopic tissue isochrones employed in prior studies: synchronized APs emerge from heterogeneous subcellular subthreshold Ca2+ signals, resembling multiscale complex processes of impulse generation within clusters of neurons in neuronal networks.

Transcriptome States Reflect Imaging of Aging States.

In this study, we describe a morphological biomarker that detects multiple discrete subpopulations (or "age-states") at several chronological ages in a population of nematodes (Caenorhabditis elegans). We determined the frequencies of three healthy adult states and the timing of the transitions between them across the lifespan. We used short-lived and long-lived strains to confirm the general applicability of the state classifier and to monitor state progression. This exploration revealed healthy and unhealthy states, the former being favored in long-lived strains and the latter showing delayed onset. Short-lived strains rapidly transitioned through the putative healthy state. We previously found that age-matched animals in different age-states have distinct transcriptome profiles. We isolated animals at the beginning and end of each identified state and performed microarray analysis (principal component analysis, relative sample to sample distance measurements, and gene set enrichment analysis). In some comparisons, chronologically identical individuals were farther apart than morphologically identical individuals isolated on different days. The age-state biomarker allowed assessment of aging in a novel manner, complementary to chronological age progression. We found hsp70 and some small heat shock protein genes are expressed later in adulthood, consistent with the proteostasis collapse model.

Predicting early symptomatic osteoarthritis in the human knee using machine learning classification of magnetic resonance images from the osteoarthritis initiative.

The purpose of this study is to evaluate the ability of a machine learning algorithm to classify in vivo magnetic resonance images (MRI) of human articular cartilage for development of osteoarthritis (OA). Sixty-eight subjects were selected from the osteoarthritis initiative (OAI) control and incidence cohorts. Progression to clinical OA was defined by the development of symptoms as quantified by the Western Ontario and McMaster Universities Arthritis (WOMAC) questionnaire 3 years after baseline evaluation. Multi-slice T2 -weighted knee images, obtained through the OAI, of these subjects were registered using a nonlinear image registration algorithm. T2 maps of cartilage from the central weight bearing slices of the medial femoral condyle were derived from the registered images using the multiple available echo times and were classified for "progression to symptomatic OA" using the machine learning tool, weighted neighbor distance using compound hierarchy of algorithms representing morphology (WND-CHRM). WND-CHRM classified the isolated T2 maps for the progression to symptomatic OA with 75% accuracy. CLINICAL SIGNIFICANCE: Machine learning algorithms applied to T2 maps have the potential to provide important prognostic information for the development of OA. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2243-2250, 2017.

CREB-binding protein levels in the rat hippocampus fail to predict chronological or cognitive aging.

Normal cognitive aging is associated with deficits in memory processes dependent on the hippocampus, along with large-scale changes in the hippocampal expression of many genes. Histone acetylation can broadly influence gene expression and has been recently linked to learning and memory. We hypothesized that CREB-binding protein (CBP), a key histone acetyltransferase, may contribute to memory decline in normal aging. Here, we quantified CBP protein levels in the hippocampus of young, aged unimpaired, and aged impaired rats, classified on the basis of spatial memory capacity documented in the Morris water maze. First, CBP-immunofluorescence was quantified across the principal cell layers of the hippocampus using both low and high resolution laser scanning imaging approaches. Second, digital images of CBP immunostaining were analyzed by a multipurpose classifier algorithm with validated sensitivity across many types of input materials. Finally, CBP protein levels in the principal subfields of the hippocampus were quantified by quantitative Western blotting. CBP levels were equivalent as a function of age and cognitive status in all analyses. The sensitivity of the techniques used was substantial, sufficient to reveal differences across the principal cell fields of the hippocampus, and to correctly classify images from young and aged animals independent of CBP immunoreactivity. The results are discussed in the context of recent evidence suggesting that CBP decreases may be most relevant in conditions of aging that, unlike normal cognitive aging, involve significant neuron loss.

Molecular characterization of the transition to mid-life in Caenorhabditis elegans.

We present an initial molecular characterization of a morphological transition between two early aging states. In previous work, an age score reflecting physiological age was developed using a machine classifier trained on images of worm populations at fixed chronological ages throughout their lifespan. The distribution of age scores identified three stable post-developmental states and transitions. The first transition occurs at day 5 post-hatching, where a significant percentage of the population exists in both state I and state II. The temperature dependence of the timing of this transition (Q 10 ~ 1.17) is too low to be explained by a stepwise process with an enzymatic or chemical rate-limiting step, potentially implicating a more complex mechanism. Individual animals at day 5 were sorted into state I and state II groups using the machine classifier and analyzed by microarray expression profiling. Despite being isogenic, grown for the same amount of time, and indistinguishable by eye, these two morphological states were confirmed to be molecularly distinct by hierarchical clustering and principal component analysis of the microarray results. These molecular differences suggest that pharynx morphology reflects the aging state of the whole organism. Our expression profiling yielded a gene set that showed significant overlap with those from three previous age-related studies and identified several genes not previously implicated in aging. A highly represented group of genes unique to this study is involved in targeted ubiquitin-mediated proteolysis, including Skp1-related (SKR), F-box-containing, and BTB motif adaptors.

Automatic detection of melanoma progression by histological analysis of secondary sites.

We present results from machine classification of melanoma biopsies sectioned and stained with hematoxylin/eosin (H&E) on tissue microarrays (TMA). The four stages of melanoma progression were represented by seven tissue types, including benign nevus, primary tumors with radial and vertical growth patterns (stage I) and four secondary metastatic tumors: subcutaneous (stage II), lymph node (stage III), gastrointestinal and soft tissue (stage IV). Our experiment setup comprised 14,208 image samples based on 164 TMA cores. In our experiments, we constructed an HE color space by digitally deconvolving the RGB images into separate H (hematoxylin) and E (eosin) channels. We also compared three different classifiers: Weighted Neighbor Distance (WND), Radial Basis Functions (RBF), and k-Nearest Neighbors (kNN). We found that the HE color space consistently outperformed other color spaces with all three classifiers, while the different classifiers did not have as large of an effect on accuracy. This showed that a more physiologically relevant representation of color can have a larger effect on correct image interpretation than downstream processing steps. We were able to correctly classify individual fields of view with an average of 96% accuracy when randomly splitting the dataset into training and test fields. We also obtained a classification accuracy of 100% when testing entire cores that were not previously used in training (four random trials with one test core for each of 7 classes, 28 tests total). Because each core corresponded to a different patient, this test more closely mimics a clinically relevant setting where new patients are evaluated based on training with previous cases. The analysis method used in this study contains no parameters or adjustments that are specific to melanoma morphology, suggesting it can be used for analyzing other tissues and phenotypes, as well as potentially different image modalities and contrast techniques.