GESTALT-Genetic and Epigenetic Signatures of Translational Aging Laboratory Testing

National Institute On Aging

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Abstract

Progress in 2021: 1- GESTALT donors recruitment is still ongoing; previously we reached 99 donors out of the 100 donors project goal and because of the pandemic there was no increase on this number for this year. In addition to the baseline visit, donors have follow-up visits in year 2 and 4. Now, we have 62 donors that completed visit 2 and 20 donors that completed visit 3. 2-Transcriptome: a. The RNA sequencing of all the isolated cell types was completed for 1400 sample from 99 donors. The cell-specific gene expression analysis was determined for the first 50 donors and are part of the manuscript Specification of human immune cell epigenetic identity by combinations of transcription factors that was accepted and will be published soon in Immunity. The age-associate changes in transcriptome are still in progress. b. The Skeletal Muscle RNA sequencing and analysis is completed, and the manuscript Skeletal Muscle Transcriptome in Healthy Aging was published in Nature Communications by Tumasian et al in April 2021. In summary, the expression levels of 57,205 protein-coding and non-coding RNAs were regressed to age by linear and negative binomial regression models adjusted for sex revealed 1,134 RNAs were significantly associated with age (p<0.01). The differentially expressed mRNAs encoded proteins implicated in diverse biological processes, including cellular senescence, insulin signaling, muscle contraction, and myogenesis. Further analyses revealed specific splice variants that changed significantly in skeletal muscle with age, including several that encoded proteins involved in oxidative phosphorylation. 3- Proteome: a. Proteomic studies of the skeletal muscle was completed and the manuscript Discovery proteomics in aging human skeletal muscle finds change in spliceosome, immunity, proteostasis and mitochondria was published in elife by Ubaida-Mohien et al. b. Proteomic studies of the different cell types was completed for around 950 sample from 99 donor and the data analysis is in progress. 4- Epigenetics a. Chromatin Accessibility: Age-associate changes in chromatin accessibility were studied in monocytes and B cells (naive and memory). Monocytes open chromatin studies were performed in 21 donors (young: 20-55year n=12, old: 60-85 years n=9). PCA analysis of differentially open chromatin (DOC) identified three major clusters, only old (n=5), only young (n=7) and old and young together (n=4 O+5 Y). This heterogeneous healthy old population was characterized by significant enrichment of DOC comprising NF-kappaB and ETS motifs. The genes associated with DOCs corresponded to the enrichment of pro-inflammatory pathways involving NF-kappaB and NOD-like receptor signaling, demonstrating heterogenous inflamm-aging process. Furthermore, the monocytes of the 5 old donors show distinct cytokine expression profile in intracellular cytokine assays. Inflammatory cytokines such as IL-6 and IL-1beta were already expressed in the baseline status. However, TNFalpha was neither expressed at the baseline nor after endotoxin activation (LPS). The current analysis of chromatin accessibility variations with DNA methylation, transcriptome, and serum cytokine levels are determined and a manuscript is in preparation. B cell open chromatin studies identified the naive and memory features of lymphocytes such as presence of OCT motif in memory specific open chromatin and ETS in naive B cells. Manuscript describing the lymphocytes differentiation properties is in preparation. b. Methylome: DNA methylation data was obtained from 76 donor and the cell specific methylation was determined. In summary our analysis identified lineage-specific signatures and their relationships to other epigenomic chromatin features. Lineage-specific sites of hypomethylation coincided with enhancer chromatin marks while lineage-specific hypermethylation sites presented only in adaptive immune cells but not the innate ones. We further studied if the cell specific methylation was related to the cell specific gene expression. Our data shows that small portion of cell specific upregulated genes were also hypomethylated in that specific cell type. The cell specific methylation and gene expression manuscript by Roy et al was accepted and will be published soon in Immunity. In addition to the cell specific methylation studies, age-associate changes in DNA methylation for the different cell types are in a manuscript preparation and almost ready for submission. 5- Serum Cytokines: pro- and anti-inflammatory cytokines were measured in the serum of 90 donors using the SimOa assay. some of the cytokines that was measured are IL-6, TNFa, IFNg, IFNa, TRAIL, IL-10, IL-8 and IL-12 p70. We observed an increase of the cytokines levels with age only for IL-6 and IFNg. Correlation of the cytokine levels with transcriptome and Methylome is ongoing.