ABSTRACT
Several studies have indicated that interrupted epigenetic reprogramming using Yamanaka transcription factors (OSKM) can rejuvenate cells from old laboratory animals and humans. However, the potential of OSKM-induced rejuvenation in brain tissue has been less explored. Here, we aimed to restore cognitive performance in 25.3-month-old female Sprague-Dawley rats using OSKM gene therapy for 39 days. Their progress was then compared with the cognitive performance of untreated 3.5-month-old rats as well as old control rats treated with a placebo adenovector. The Barnes maze test, used to assess cognitive performance, demonstrated enhanced cognitive abilities in old rats treated with OSKM compared to old control animals. In the treated old rats, there was a noticeable trend towards improved spatial memory relative to the old controls. Further, OSKM gene expression did not lead to any pathological alterations within the 39 days. Analysis of DNA methylation following OSKM treatment yielded three insights. First, epigenetic clocks for rats suggested a marginally significant epigenetic rejuvenation. Second, chromatin state analysis revealed that OSKM treatment rejuvenated the methylome of the hippocampus. Third, an epigenome-wide association analysis indicated that OSKM expression in the hippocampus of old rats partially reversed the age-related increase in methylation. In summary, the administration of Yamanaka genes via viral vectors rejuvenates the functional capabilities and the epigenetic landscape of the rat hippocampus.
ABSTRACT
There is converging evidence that young blood conveys cells, vesicles, and molecules able to revitalize function and restore organ integrity in old individuals. We assessed the effects of young plasma on the lifespan, epigenetic age, and healthspan of old female rats. Beginning at 25.6 months of age, a group of 9 rats (group T) was intraperitoneally injected with plasma from young rats until their natural death. A group of 8 control rats of the same age received no treatment (group C). Blood samples were collected every other week. Survival curves showed that from age 26 to 30 months, none of the group T animals died, whereas the survival curve of group C rats began to decline at age 26 months. Blood DNAm age versus chronological age showed that DNAm age in young animals increased faster than chronological age, then slowed down, entering a plateau after 27 months. The DNAm age of the treated rats fell below the DNAm age of controls and, in numerical terms, remained consistently lower until natural death. When rats were grouped according to the similarities in their differential blood DNA methylation profile, samples from the treated and control rats clustered in separate groups. Analysis of promoter differential methylation in genes involved in systemic regulatory activities revealed specific GO term enrichment related to the insulin-like factors pathways as well as to cytokines and chemokines associated with immune and homeostatic functions. We conclude that young plasma therapy may constitute a natural, noninvasive intervention for epigenetic rejuvenation and health enhancement.
Subject(s)
Longevity , Physical Appearance, Body , Female , Rats , Animals , Longevity/genetics , DNA Methylation , Aging/genetics , Epigenesis, GeneticABSTRACT
Young blood plasma is known to confer beneficial effects on various organs in mice and rats. However, it was not known whether plasma from young adult pigs rejuvenates old rat tissues at the epigenetic level; whether it alters the epigenetic clock, which is a highly accurate molecular biomarker of aging. To address this question, we developed and validated six different epigenetic clocks for rat tissues that are based on DNA methylation values derived from n = 613 tissue samples. As indicated by their respective names, the rat pan-tissue clock can be applied to DNA methylation profiles from all rat tissues, while the rat brain, liver, and blood clocks apply to the corresponding tissue types. We also developed two epigenetic clocks that apply to both human and rat tissues by adding n = 1366 human tissue samples to the training data. We employed these six rat clocks to investigate the rejuvenation effects of a porcine plasma fraction treatment in different rat tissues. The treatment more than halved the epigenetic ages of blood, heart, and liver tissue. A less pronounced, but statistically significant, rejuvenation effect could be observed in the hypothalamus. The treatment was accompanied by progressive improvement in the function of these organs as ascertained through numerous biochemical/physiological biomarkers, behavioral responses encompassing cognitive functions. An immunoglobulin G (IgG) N-glycosylation pattern shift from pro- to anti-inflammatory also indicated reversal of glycan aging. Overall, this study demonstrates that a young porcine plasma-derived treatment markedly reverses aging in rats according to epigenetic clocks, IgG glycans, and other biomarkers of aging.
Subject(s)
Aging , Epigenesis, Genetic , Humans , Rats , Mice , Animals , Swine , Aging/physiology , Biomarkers , Plasma , Immunoglobulin GABSTRACT
Young blood plasma is known to confer beneficial effects on various organs in mice and rats. However, it was not known whether plasma from young pigs rejuvenates old rat tissues at the epigenetic level; whether it alters the epigenetic clock, which is a highly accurate molecular biomarker of aging. To address this question, we developed and validated six different epigenetic clocks for rat tissues that are based on DNA methylation values derived from n=613 tissue samples. As indicated by their respective names, the rat pan-tissue clock can be applied to DNA methylation profiles from all rat tissues, while the rat brain-, liver-, and blood clocks apply to the corresponding tissue types. We also developed two epigenetic clocks that apply to both human and rat tissues by adding n=1366 human tissue samples to the training data. We employed these six rat clocks to investigate the rejuvenation effects of a porcine plasma fraction treatment in different rat tissues. The treatment more than halved the epigenetic ages of blood, heart, and liver tissue. A less pronounced, but statistically significant, rejuvenation effect could be observed in the hypothalamus. The treatment was accompanied by progressive improvement in the function of these organs as ascertained through numerous biochemical/physiological biomarkers and behavioral responses to assess cognitive functions. An immunoglobulin G (IgG) N-glycosylation pattern shift from pro- to anti-inflammatory also indicated reversal of glycan aging. Overall, this study demonstrates that a young porcine plasma-derived treatment markedly reverses aging in rats according to epigenetic clocks, IgG glycans, and other biomarkers of aging.
ABSTRACT
Using DNA methylation profiles (n = 15,456) from 348 mammalian species, we constructed phyloepigenetic trees that bear marked similarities to traditional phylogenetic ones. Using unsupervised clustering across all samples, we identified 55 distinct cytosine modules, of which 30 are related to traits such as maximum life span, adult weight, age, sex, and human mortality risk. Maximum life span is associated with methylation levels in HOXL subclass homeobox genes and developmental processes and is potentially regulated by pluripotency transcription factors. The methylation state of some modules responds to perturbations such as caloric restriction, ablation of growth hormone receptors, consumption of high-fat diets, and expression of Yamanaka factors. This study reveals an intertwined evolution of the genome and epigenome that mediates the biological characteristics and traits of different mammalian species.
Subject(s)
DNA Methylation , Epigenesis, Genetic , Mammals , Adult , Animals , Humans , Epigenome , Genome , Mammals/genetics , PhylogenyABSTRACT
Ageing is associated with impaired performance in recognition memory, a process that consists of the discrimination of familiar and novel stimuli. Previous studies have shown the impact of ageing on object recognition memories. However, the early stages of memory impairment remain unknown. To fill this gap, we aimed at evaluating the ability of young (Y), middle-aged (MA), and senile (S) female Sprague-Dawley rats to retain 24 h long-term recognition memory. The MA cohort was included to characterise early memory deficits under two behavioural paradigms based on spontaneous location recognition (SLR) and spontaneous object recognition (SOR) tasks. In the SLR task, there was a markedly diminished novel discrimination capacity in the MA and S rats compared with the Y ones. In the SOR task, S rats evidenced a deterioration in novelty discrimination, while MA rats partially preserved the capacity to distinguish the new stimulus as compared with Y rats. Regarding early changes from MA to S rats, immunohistochemistry showed a marked decrease in the number and diameter of adult-born immature neurons in the Dentate Gyrus (DG) with a positive correlation with behavioural performance in the SLR task. Furthermore, we found a slight reduction in CA3 mature neurons and a decrease in the number of total microglia in the perirhinal cortex (Prh) in MA and S rats as compared with Y rats. As regards changes that were only observed in S rats, we found an increase in the number of total and reactive microglia in CA3 and a reduction in the number of total microglia in the DG. We conclude that spatial discrimination capacity could be affected earlier than feature discrimination capacity. We suggest that early depletion of neurogenesis in MA rats is involved in object location recognition deficits, whereas the disruption of microglial homeostasis in the Prh could be associated with object feature discrimination capacity.
Subject(s)
Perirhinal Cortex , Animals , Exploratory Behavior/physiology , Female , Hippocampus/physiology , Humans , Rats , Rats, Sprague-Dawley , Recognition, Psychology/physiologyABSTRACT
In humans and rats, aging is associated with a progressive deterioration of spatial learning and memory. These functional alterations are correlated with morphological and molecular changes in the hippocampus. Here, we assessed age-related changes in DNA methylation (DNAm) landscape in the rat hippocampus and the correlation of spatial memory with hippocampal DNAm age in 2.6- and 26.6-month-old rats. Spatial memory performance was assessed with the Barnes maze test. To evaluate learning ability and spatial memory retention, we assessed the time spent by animals in goal sector 1 (GS1) and 3 (GS3) when the escape box was removed. The rat pan-tissue clock was applied to DNAm data from hippocampal tissue. An enrichment pathway analysis revealed that neuron fate commitment, brain development, and central nervous system development were processes whose underlying genes were enriched in hypermethylated CpGs in the old rats. In the old rat hippocampi, the methylation levels of CpG proximal to transcription factors associated with genes Pax5, Lbx1, Nr2f2, Hnf1b, Zic1, Zic4, Hoxd9; Hoxd10, Gli3, Gsx1 and Lmx1b, and Nipbl showed a significant regression with spatial memory performance. Regression analysis of different memory performance indices with hippocampal DNAm age was significant. These results suggest that age-related hypermethylation of transcription factors related to certain gene families, such as Zic and Gli, may play a causal role in the decline in spatial memory in old rats. Hippocampal DNAm age seems to be a reliable index of spatial memory performance in young and old rats.
Subject(s)
DNA Methylation , Spatial Memory , Animals , Rats , Aging/genetics , Cell Cycle Proteins/genetics , Epigenesis, Genetic , Hippocampus , Maze Learning/physiology , Spatial Memory/physiology , Transcription Factors/geneticsABSTRACT
Hippocampus serves as a pivotal role in cognitive and emotional processes, as well as in the regulation of the hypothalamus-pituitary axis. It is known to undergo mild neurodegenerative changes during normal aging and severe atrophy in Alzheimer's disease. Furthermore, dysregulation in the hippocampal function leads to epilepsy and mood disorders. In the first section, we summarized the most salient knowledge on the role of glial cell-line-derived neurotrophic factor and its receptors focused on aging, cognition and neurodegenerative and hippocampal-related neurological diseases mentioned above. In the second section, we reviewed the therapeutic approaches, particularly gene therapy, using glial cell-line-derived neurotrophic factor or its gene, as a key molecule in the development of neurological disorders. In the third section, we pointed at the potential of regenerative medicine, as an emerging and less explored strategy for the treatment of hippocampal disorders. We briefly reviewed the use of partial reprogramming to restore brain functions, non-neuronal cell reprogramming to generate neural stem cells, and neural progenitor cells as source-specific neuronal types to be implanted in animal models of specific neurodegenerative disorders.
ABSTRACT
The view of aging has evolved in parallel with the advances in biomedical sciences. Long considered as an irreversible process where interventions were only aimed at slowing down its progression, breakthrough discoveries like animal cloning and cell reprogramming have deeply changed our understanding of postnatal development, giving rise to the emerging view that the epigenome is the driver of aging. The idea was significantly strengthened by the converging discovery that DNA methylation (DNAm) at specific CpG sites could be used as a highly accurate biomarker of age defined by an algorithm known as the Horvath clock. It was at this point where epigenetic rejuvenation came into play as a strategy to reveal to what extent biological age can be set back by making the clock tick backwards. Initial evidence suggests that when the clock is forced to tick backwards in vivo, it is only able to drag the phenotype to a partially rejuvenated condition. In order to explain the results, a bimodular epigenome is proposed, where module A represents the DNAm clock component and module B the remainder of the epigenome. Epigenetic rejuvenation seems to hold the key to arresting or even reversing organismal aging.
Subject(s)
Aging/genetics , Epigenome/genetics , Rejuvenation/physiology , Animals , Biomarkers , Cellular Reprogramming , DNA Methylation , HumansABSTRACT
Regulatory mechanisms range from a single level of control in simple metazoans to multi-level hierarchical control networks in higher animals. Organismal regulation encompasses homeostatic and circadian networks that are interconnected, with no documented exceptions. The epigenetic clock is a highly accurate biomarker of age in humans, defined by a mathematical algorithm based on the methylation of a subset of age-related CpG sites on DNA. Experimental evidence suggests the existence of an underlying regulatory mechanism. By analogy with other integrative systems as the neuroendocrine-immune network and the circadian clocks, a hierarchical organization in the control of the ticking rate of the epigenetic clock is hypothesized here. The hierarchical organization of the neuroendocrine, immune and circadian systems is briefly reviewed. This is followed by a brief review of the epigenetic clock at cell level. Finally, different lines of indirect evidence, consistent with the existence of a central pacemaker controlling the ticking rate of the epigenetic clock at organismal level are discussed. The concluding remarks put the hierarchical model proposed for the control of the clock into an evolutionary perspective. Within this perspective, the present hypothesis is intended as a conceptual outline based on designs consistently favored by evolution in higher animals.
Subject(s)
DNA Methylation , Epigenomics , Aging/genetics , Animals , Circadian Rhythm/genetics , Epigenesis, Genetic , Humans , Mammals/geneticsABSTRACT
BACKGROUND: Conventional cell reprogramming involves converting a somatic cell line into induced pluripotent stem cells (iPSC), which subsequently can be re-differentiated to specific somatic cell types. Alternatively, partial cell reprogramming converts somatic cells into other somatic cell types by transient expression of pluripotency genes thus generating intermediates that retain their original cell identity, but are responsive to appropriate cocktails of specific differentiation factors. Additionally, biological rejuvenation by partial cell reprogramming is an emerging avenue of research. OBJECTIVE: Here, we will briefly review the emerging information pointing to partial reprogramming as a suitable strategy to achieve cell reprogramming and rejuvenation, bypassing cell dedifferentiation. METHODS: In this context, regulatable pluripotency gene expression systems are the most widely used at present to implement partial cell reprogramming. For instance, we have constructed a regulatable bidirectional adenovector expressing Green Fluorescent Protein and oct4, sox2, klf4 and c-myc genes (known as the Yamanaka genes or OSKM). RESULTS: Partial cell reprogramming has been used to reprogram fibroblasts to cardiomyocytes, neural progenitors and neural stem cells. Rejuvenation by cyclic partial reprogramming has been achieved both in vivo and in cell culture using transgenic mice and cells expressing the OSKM genes, respectively, controlled by a regulatable promoter. CONCLUSION: Partial reprogramming emerges as a powerful tool for the genesis of iPSC-free induced somatic cells of therapeutic value and for the implementation of in vitro and in vivo rejuvenation keeping cell type identity unchanged.
Subject(s)
Cell Differentiation , Cellular Reprogramming , Pluripotent Stem Cells/cytology , Regenerative Medicine , Rejuvenation/physiology , Animals , Humans , Kruppel-Like Factor 4ABSTRACT
Biological rejuvenation by partial cell reprogramming is an emerging avenue of research. In this context, regulatable pluripotency gene expression systems are the most widely used at present. We have constructed a regulatable bidirectional adenovector expressing the humanized green fluorescent protein (GFP) and oct4, sox2, klf4, and c-myc genes (known as the Yamanaka genes or OSKM). The OSKM genes are arranged as a bicistronic tandem (hSTEMCCA tandem), which is under the control of a Tet-Off bidirectional promoter that also controls the expression of the gFP gene. Separately, a constitutive cassette expresses the regulatory protein tTA. Vector DNA was transfected in HEK293 Cre cells, which were additionally infected with the helper adenovector H14, unable to package its DNA due to the Cre recombinase produced by the HEK293 Cre cells. The newly generated vector was expanded by six iterated coinfections of the above cells which were lysed at the end of the process and the adenovector purified by ultracentrifugation in a CsCl gradient. The titer of the initial preparation was 1.2 × 1012 physical viral particles/ml. As expected, GFP fluorescence in vector-transduced rat fibroblast cultures declined with the dose of doxycycline (DOX) present in the medium. Immunocytochemical analysis of transduced cells confirmed the expression of the four Yamanaka genes. Additionally, 3 days after vector injection in the hypothalamus of rats, a significant level of fluorescence was observed in the region. Addition of 2 mg/ml DOX to the drinking water reduced the GFP expression. This adenovector constitutes a promising tool for implementing nonintegrative partial cell reprogramming.
Subject(s)
Brain/physiology , Genetic Therapy/methods , Kruppel-Like Transcription Factors/genetics , Octamer Transcription Factor-3/genetics , Proto-Oncogene Proteins c-myc/genetics , Regeneration , SOXB1 Transcription Factors/genetics , Adenoviridae/genetics , Animals , Cells, Cultured , Female , Gene Transfer Techniques , Genetic Vectors/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/metabolism , Octamer Transcription Factor-3/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Rats , Rats, Sprague-Dawley , SOXB1 Transcription Factors/metabolismABSTRACT
The discovery of animal cloning and subsequent development of cell reprogramming technology were quantum leaps as they led to the achievement of rejuvenation by cell reprogramming and the emerging view that aging is a reversible epigenetic process. Here, we will first summarize the experimental achievements over the last 7 years in cell and animal rejuvenation. Then, a comparison will be made between the principles of the cumulative DNA damage theory of aging and the basic facts underlying the epigenetic model of aging, including Horvath's epigenetic clock. The third part will apply both models to two natural processes, namely, the setting of the aging clock in the mammalian zygote and the changes in the aging clock along successive generations in mammals. The first study demonstrating that skin fibroblasts from healthy centenarians can be rejuvenated by cell reprogramming was published in 2011 and will be discussed in some detail. Other cell rejuvenation studies in old humans and rodents published afterwards will be very briefly mentioned. The only in vivo study reporting that a number of organs of old progeric mice can be rejuvenated by cyclic partial reprogramming will also be described in some detail. The cumulative DNA damage theory of aging postulates that as an animal ages, toxic reactive oxygen species generated as byproducts of the mitochondria during respiration induce a random and progressive damage in genes thus leading cells to a progressive functional decline. The epigenetic model of aging postulates that there are epigenetic marks of aging that increase with age, leading to a progressive derepression of DNA which in turn causes deregulated expression of genes that disrupt cell function. The cumulative DNA damage model of aging fails to explain the resetting of the aging clock at the time of conception as well as the continued vitality of species as millenia go by. In contrast, the epigenetic model of aging straightforwardly explains both biologic phenomena. A plausible initial application of rejuvenation in vivo would be preventing adult individuals from aging thus eliminating a major risk factor for end of life pathologies. Further, it may allow the gradual achievement of whole body rejuvenation.