Progress in 2019: 1-GESTALT donors recruitment is still ongoing; in this year we reached 96 donors out of the 100 donors the project goal. 15 cell type is isolated from each donor. Four donors were scheduled for the next coming two months and with that the 100 donors will be completed. 2-Transcriptome: a. The RNA sequencing is completed on cells of 76 donors (15 cell x 76 donor = 1140 sample). b. The analysis is still in progress but in summary We began by examining to what extent transcript data obtained by RNAseq distinguish subsets of T Cells and B Cells. For T cells, we used the top 500 transcripts, and for the B cells, the top 100 transcripts ranked by variance. Although Principal Components 1 and 2 explain less than 30% of the total variance in both analyses, the separation between subclasses of T and B cells is remarkable. Next, we analyzed whether particular transcripts in specific cell types are related to age. Many age-associated transcripts were identified in all cell types (except natural killer cells) but particularly strong for muscle and granulocytes. Remarkably, the most diluted signals were for peripheral blood mononuclear cells (PBMCs) and whole blood, which are the most frequent sources of RNA in human studies. The data revealed many upregulated and down regulated mRNAs with aging across multiple cell types. Our top signal was for ATRX (Alpha thalassemia/Mental retardation syndrome X linked) mRNA, which is the most remarkable example. Expression of this gene increases progressively with aging in all cell types investigated. Regression lines drawn separately for all cell type have slopes significantly different from 0 (nominal p-value for the overall trend: <10-28), not significantly different from each other, and R2 ranging from 0.43 to 0.66 (overall R2: 0.70), in spite of the small sample size (n=25). ATRX is particularly interesting in aging research for several reasons: 1) ATRX is an ATP-driven DNA translocase belonging to the Swi/Snf family of chromatin remodelers. Diminished ATRX activity causes a rare developmental disorder characterized by severe cognitive deficit and -thalassemia. Decreasing levels of ATRX by RNA interference induces spindle defects, as well as chromosomal malalignment and segregation problems during mitosis and meiosis, unstable telomeres and predisposition to cell senescence; 2) It has been recently reported in a mouse model that ATRX deficiency induces telomere dysfunction, endocrine defects, and reduced life span; 3) The map of expression level of the ATRX gene is significantly correlated with the distribution of Beta-Amyloid in Alzheimers disease (unpublished data). c. The Skeletal Muscle RNA sequencing is completed, and a manuscript is under review in Nature Communications journal. 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 skeletal muscle are completed and a paper in title Physical Activity Associated Proteomics of Skeletal Muscle: Being Physically Active in Daily Life May Protect Skeletal Muscle From Aging was published for Ubaida-Mohien et al in Frontiers in Physiology and another manuscript is in revision after being reviewed in eLife. In summary at FDR <0.01 we identified 2,564 proteins across all 5 TMT sets. In 1,190 we had a signal strong enough for quantitative analyses. Partial least-squares regression identified three principal components that explained 37% of the variance and perfectly separated samples for older (50+ years) and younger (2049 years) participants. A complexity of studying proteomics in skeletal muscle is the heterogeneity of fiber types in skeletal muscle (type I are slow; type IIa are fast; and type IIb are very fast), which may potentially confound the comparison between different age groups. A strategy to partially address this issue is to adjust the analysis by fiber-type-specific myosin isoforms ratio (Myosin VII for Type I fiber and Myosin II for type IIa fibers), used as proxy measure of fiber-types ratio. In contrast to what has been reported in the literature, in our sample of very healthy participants, we found that the estimated ratio between type I and type II fibers declined with aging. These findings were confirmed by using both Troponin-specific isoforms or quantifying the relative abundance of type I and IIa fibers in muscle microscopic images in a separate sample. A second important confounder is the level of physical activity (evaluated by self-report), which declines with aging and also strongly affects muscle tissue. To address this issue, we analyzed the effect of self-reported physical activity (on a 5-point scale) on muscle proteins adjusting for age, sex, fiber type ratio, and TMT set. We found 159 proteins associated with physical activity with a p-value <0.05 and adjusted beta coefficient >0.01 or <0.01. Of these, 79 were downregulated and 77 were upregulated. More than 90% of upregulated proteins are mitochondrial, either involved in oxidative phosphorylation or in the Kreb Cycle metabolism. Based on these findings, analyses looking at the effect of age were all adjusted for self-reported physical activity. In the age analysis, we found 103 proteins that were significantly upregulated or downregulated with age. Contrary to the previous analysis, almost 50% of the proteins downregulated with age are mitochondrial proteins. Interestingly, many of these are related to the ubiquinone complex. Moreover, the protein Monocarboxylate transporter 1 (Lactate transporter) is significantly downregulated. Although knowledge of this protein is still limited, a deficiency may result in an acidic intracellular environment created by muscle activity with consequent degeneration of muscle and release of myoglobin and creatine kinase. It has been shown that this defect might compromise extreme performance in otherwise healthy individuals. b- Proteomic studies of the blood cells are completed for 56 donors (around 700 sample) and the analysis is in progress. 4- Monocyte samples for ATACSeq were collected from 30 donors of wide age range (20 80 years old), library preparation and sequencing were completed. The analysis is still in progress but in summary it revealed selective activation of pro-inflammatory pathways involving many signaling pathways in monocytes from old individuals compared to the young ones. Relationships of these chromatin accessibility changes to transcriptome are ongoing.

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National Institute on Aging (NIA)
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Gonzalez-Freire, Marta; Semba, Richard D; Ubaida-Mohien, Ceereena et al. (2017) The Human Skeletal Muscle Proteome Project: a reappraisal of the current literature. J Cachexia Sarcopenia Muscle 8:5-18
Levine, Morgan E; Lu, Ake T; Chen, Brian H et al. (2016) Menopause accelerates biological aging. Proc Natl Acad Sci U S A 113:9327-32